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U-insertion/deletion (U-indel) RNA editing in trypanosome mitochondria is directed by guide RNAs (gRNAs). This editing may developmentally control respiration in bloodstream forms (BSF) and insect procyclic forms (PCF). Holo-editosomes include the accessory RNA Editing Substrate Binding Complex (RESC) and RNA Editing Helicase 2 Complex (REH2C), but the specific proteins controlling differential editing remain unknown. Also, RNA editing appears highly error prone because most U-indels do not match the canonical pattern. However, despite extensive non-canonical editing of unknown functions, accurate canonical editing is required for normal cell growth. In PCF, REH2C controls editing fidelity in RESC-bound mRNAs. Here, we report that KREH2, a REH2C-associated helicase, developmentally controls programmed non-canonical editing, including an abundant 3′ element in ATPase subunit 6 (A6) mRNA. The 3′ element sequence is directed by a proposed novel regulatory gRNA. In PCF, KREH2 RNAi-knockdown up-regulates the 3′ element, which establishes a stable structure hindering element removal by canonical initiator-gRNA-directed editing. In BSF, KREH2-knockdown does not up-regulate the 3′ element but reduces its high abundance. Thus, KREH2 differentially controls extensive non-canonical editing and associated RNA structure via a novel regulatory gRNA, potentially hijacking factors as a ‘molecular sponge’. Furthermore, this gRNA is bifunctional, serving in canonical CR4 mRNA editing whilst installing a structural element in A6 mRNA.

Mitochondrial DNA of protists of order Kinetoplastida comprises thousands of interlinked circular molecules arranged in a network. There are two types of molecules called minicircles and maxicircles. Minicircles encode guide RNA (gRNA) genes whose transcripts mediate post-transcriptional editing of maxicircle encoded genes. Minicircles are diverse. The human sleeping sickness parasite Trypanosoma brucei has one of the most diverse sets of minicircle classes of all studied trypanosomatids with hundreds of different classes, each encoding one to four genes mainly within cassettes framed by 18 bp inverted repeats. A third of cassettes have no identifiable gRNA genes even though their sequence structures are similar to cassettes with identifiable genes. Only recently have almost all minicircle classes for some subspecies and isolates of T. brucei been sequenced and annotated with corresponding verification of gRNA expression by small-RNA transcriptome data. These data sets provide a rich resource for understanding the structure of minicircle classes, cassettes and gRNA genes and their transcription. Here, we provide a statistical description of the functionality, expression status, structure and sequence of gRNA genes in a differentiation-competent, laboratory-adapted strain of T. brucei. We obtain a clearer definition of what is a gRNA gene. Our analysis supports the idea that many, if not all, cassettes without an identifiable gRNA gene contain decaying remnants of once functional gRNA genes. Finally, we report several new, unexplained discoveries such as the association between cassette position on the minicircle and gene expression and functionality, and the association between gene initiation sequence and anchor position.

Purpose Surra is an economically important livestock disease in many low- and middle-income countries, including those of Northern Africa. The disease is caused by the biting fly-transmitted subspecies Trypanosoma brucei evansi, which is very closely related to the tsetse-transmitted subspecies T. b. brucei and the sexually transmitted subspecies T. b. equiperdum. At least two phylogenetically distinct groups of T. b. evansi can be distinguished, called type A and type B. These evolved from T. b. brucei independently. The close relationships between the T. brucei subspecies and the multiple evolutionary origins of T. b. evansi pose diagnostic challenges. Methods Here we use previously established and newly developed PCR assays based on nuclear and mitochondrial genetic markers to type the causative agent of recent trypanosome infections of camels in Southern Algeria. Results/conclusion We confirm that these infections have been caused by T. b. evansi type A. We also report a newly designed PCR assay specific for T. b. evansi type A that we expect will be of diagnostic use for the community.

The World Health Organization targeted

Kinetoplastid parasites cause diverse neglected diseases in humans and livestock, with an urgent need for new treatments. Survival of kinetoplastids depends on their uniquely structured mitochondrial genome (kDNA), the eponymous kinetoplast. Here we report development of a high-content screen for pharmacologically induced kDNA loss, based on specific staining of parasites and automated image analysis. As proof-of-concept we screened a diverse set of ∼14,000 small molecules and exemplify a validated hit as a novel kDNA-targeting compound.

The World Health Organization targeted Trypanosoma brucei gambiense (Tbg) human African trypanosomiasis for elimination of transmission by 2030. Sensitive molecular markers that specifically detect Tbg type 1 (Tbg1) parasites will be important tools to assist in reaching this goal. Here, we aim at improving molecular diagnosis of Tbg1 infections by targeting the abundant mitochondrial minicircles within the kinetoplast of Trypanosoma brucei parasites. Using Next-Generation Sequencing of total cellular DNA extracts, we assembled and annotated the kinetoplast genome and investigated minicircle sequence diversity in 38 animal- and human-infective trypanosome strains. Computational analyses recognized a total of 241 Minicircle Sequence Classes as Tbg1-specific, of which three were shared by the 18 studied Tbg1 strains. We then developed a novel multiplex quantitative PCR assay (g-qPCR3) targeting one Tbg1-specific minicircle and three Tbg1-specific or Trypanozoon-specific markers. Molecular analyses revealed that the minicircle-based assay is applicable on animals and is as specific as the TgsGP-based assay, the current golden standard for molecular detection of Tbg1. The median copy number of the targeted minicircle was equal to eight, suggesting that our minicircle-based assay may be used for the sensitive detection of Tbg1 parasites. Finally, annotation of the targeted minicircle sequence indicated that it encodes genes essential for the survival of the parasite, and will thus likely be preserved in natural Tbg1 populations. These results demonstrated that our minicircle-based assay is a promising new tool for reliable and sensitive detection of Tbg1 infections in humans and animals.

Mitochondrial DNA of protists of order

Animal African Trypanosomiasis (AAT) is a debilitating livestock disease prevalent across sub-Saharan Africa, a main cause of which is the protozoan parasite Trypanosoma congolense. In comparison to the well-studied T. brucei, there is a major paucity of knowledge regarding the biology of T. congolense. Here, we use a combination of omics technologies and novel genetic tools to characterise core metabolism in T. congolense mammalian-infective bloodstream-form parasites, and test whether metabolic differences compared to T. brucei impact upon sensitivity to metabolic inhibition. Like the bloodstream stage of T. brucei, glycolysis plays a major part in T. congolense energy metabolism. However, the rate of glucose uptake is significantly lower in bloodstream stage T. congolense, with cells remaining viable when cultured in concentrations as low as 2 mM. Instead of pyruvate, the primary glycolytic endpoints are succinate, malate and acetate. Transcriptomics analysis showed higher levels of transcripts associated with the mitochondrial pyruvate dehydrogenase complex, acetate generation, and the glycosomal succinate shunt in T. congolense, compared to T. brucei. Stable-isotope labelling of glucose enabled the comparison of carbon usage between T. brucei and T. congolense, highlighting differences in nucleotide and saturated fatty acid metabolism. To validate the metabolic similarities and differences, both species were treated with metabolic inhibitors, confirming that electron transport chain activity is not essential in T. congolense. However, the parasite exhibits increased sensitivity to inhibition of mitochondrial pyruvate import, compared to T. brucei. Strikingly, T. congolense exhibited significant resistance to inhibitors of fatty acid synthesis, including a 780-fold higher EC50 for the lipase and fatty acid synthase inhibitor Orlistat, compared to T. brucei. These data highlight that bloodstream form T. congolense diverges from T. brucei in key areas of metabolism, with several features that are intermediate between bloodstream- and insect-stage T. brucei. These results have implications for drug development, mechanisms of drug resistance and host-pathogen interactions., Author summary Animal African Trypanosomiasis (AAT), also known as Nagana, is a devastating disease affecting livestock across sub-Saharan Africa. AAT is primarily caused by the parasite Trypanosoma congolense, yet our biological knowledge about this pathogen is poor, especially compared to the related species T. brucei, subspecies of which cause the human disease Sleeping Sickness. Understanding the core metabolism of T. congolense is crucial in order to gain insights into the infection biology of this important pathogen, as well as providing the potential to identify new drug targets. In this work, we addressed the lack of knowledge concerning T. congolense by carrying out a comprehensive analysis of core metabolism, and comparing the data to T. brucei. We then used the findings of metabolic differences to predict differential sensitivity to inhibitors of metabolic function. We show that unlike T. brucei, where glucose metabolism leads to high levels of pyruvate excretion, T. congolense metabolises glucose to other end-products, namely succinate, malate and acetate. Moreover, there are pronounced differences in the way T. congolense uses glucose to feed into other areas of metabolism. Further analysis also suggests that T. congolense mostly scavenges lipids and fatty acids, rather than synthesising them de novo. To validate these findings, we confirm that T. congolense is differentially susceptible to metabolic inhibitors compared to T. brucei, and that, in particular, T. congolense is significantly less sensitive to inhibitors of fatty acid synthesis. Our study provides a foundation of functional metabolic knowledge on T. congolense, with insights into how this parasite fundamentally differs from T. brucei.

The single-celled parasite Trypanosoma brucei causes sleeping sickness in humans and nagana in livestock and is transmitted by hematophagous tsetse flies. Lifecycle progression from mammalian bloodstream form to tsetse midgut form and, subsequently, infective salivary gland form depends on complex developmental steps and migration within different fly tissues. As the parasite colonises the glucose-poor insect midgut, its ATP production is thought to depend on activation of mitochondrial amino acid catabolism via oxidative phosphorylation. This process involves respiratory chain complexes and the F1FO-ATP synthase, and it requires protein subunits of these complexes that are encoded in the parasite’s mitochondrial DNA (kinetoplast or kDNA). Here we show that a progressive loss of kDNA-encoded functions correlates with an increasingly impaired ability of T. brucei to initiate and complete its development in the tsetse. First, parasites with a mutated F1FO-ATP synthase with a reduced capacity for oxidative phosphorylation can initiate differentiation from bloodstream to insect form, but they are unable to proliferate in vitro. Unexpectedly, these cells can still colonise the tsetse midgut. However, these parasites exhibit a motility defect and are severely impaired in colonising or migrating to subsequent tsetse tissues. Second, parasites with a fully disrupted F1FO-ATP synthase complex that is completely unable to produce ATP by oxidative phosphorylation can still differentiate to the first insect stage in vitro but die within a few days and cannot establish a midgut infection in vivo. Third, mutant parasites lacking kDNA entirely can initiate differentiation but die within 24 h. Together, these three scenarios show that efficient ATP production via oxidative phosphorylation is not essential for initial colonisation of the tsetse vector, but it is required to power trypanosome migration within the fly.

Kinetoplastids are protists defined by one of the most complex mitochondrial genomes in nature, the kinetoplast. In the sleeping sickness parasite Trypanosoma brucei, the kinetoplast is a chain mail-like network of two types of interlocked DNA molecules: a few dozen ∼23-kb maxicircles (homologs of the mitochondrial genome of other eukaryotes) and thousands of ∼1-kb minicircles. Maxicircles encode components of respiratory chain complexes and the mitoribosome. Several maxicircle-encoded mRNAs undergo extensive post-transcriptional RNA editing via addition and deletion of uridines. The process is mediated by hundreds of species of minicircle-encoded guide RNAs (gRNAs), but the precise number of minicircle classes and gRNA genes was unknown. Here we present the first essentially complete assembly and annotation of the kinetoplast genome of T. brucei. We have identified 391 minicircles, encoding not only ∼930 predicted ‘canonical’ gRNA genes that cover nearly all known editing events (accessible via the web at http://hank.bio.ed.ac.uk), but also ∼370 ‘non-canonical’ gRNA genes of unknown function. Small RNA transcriptome data confirmed expression of the majority of both categories of gRNAs. Finally, we have used our data set to refine definitions for minicircle structure and to explore dynamics of minicircle copy numbers.

Equine trypanosomosis is a complex of infectious diseases called dourine, nagana and surra. It is caused by several species of the genus Trypanosoma that are transmitted cyclically by tsetse flies, mechanically by other haematophagous flies, or sexually. Trypanosoma congolense (subgenus Nannomonas) and T. vivax (subgenus Dutonella) are genetically and morphologically distinct from T. brucei, T. equiperdum and T. evansi (subgenus Trypanozoon). It remains controversial whether the three latter taxa should be considered distinct species. Recent outbreaks of surra and dourine in Europe illustrate the risk and consequences of importation of equine trypanosomosis with infected animals into non-endemic countries. Knowledge on the epidemiological situation is fragmentary since many endemic countries do not report the diseases to the World Organisation for Animal Health, OIE. Other major obstacles to the control of equine trypanosomosis are the lack of vaccines, the inability of drugs to cure the neurological stage of the disease, the inconsistent case definition and the limitations of current diagnostics. Especially in view of the ever-increasing movement of horses around the globe, there is not only the obvious need for reliable curative and prophylactic drugs but also for accurate diagnostic tests and algorithms. Unfortunately, clinical signs are not pathognomonic, parasitological tests are not sufficiently sensitive, serological tests miss sensitivity or specificity, and molecular tests cannot distinguish the taxa within the Trypanozoon subgenus. To address the limitations of the current diagnostics for equine trypanosomosis, we recommend studies into improved molecular and serological tests with the highest possible sensitivity and specificity. We realise that this is an ambitious goal, but it is dictated by needs at the point of care. However, depending on available treatment options, it may not always be necessary to identify which trypanosome taxon is responsible for a given infection.

Animal African Trypanosomiasis (AAT) is a debilitating livestock disease prevalent across sub-Saharan Africa, a main cause of which is the protozoan parasiteTrypanosoma congolense. In comparison to the well-studiedT. brucei, there is a major paucity of knowledge regarding the biology ofT. congolense. Here, we use a combination of omics technologies and novel genetic tools to characterise core metabolism inT. congolensemammalian-infective bloodstream-form parasites, and test whether metabolic differences compared toT. bruceiimpact upon drug sensitivity. LikeT. brucei, glycolysis plays a major part inT. congolenseenergy metabolism. However, the rate of glucose uptake is significantly reduced inT. congolense, with cells remaining viable when cultured in concentrations as low as 2 mM. Instead of pyruvate, the primary glycolytic endpoints are succinate, malate and acetate. Comparative transcriptomics analysis showed higher levels of activity associated with the mitochondrial pyruvate dehydrogenase complex, acetate generation and the succinate shunt inT. congolense. However, based on omics analysis and chemical inhibition, there does not appear to be significant levels of oxidative phosphorylation. Stable-isotope labelling of glucose enabled the comparison of carbon usage betweenT. bruceiandT. congolense, highlighting differences in nucleotide and fatty acid metabolism. To validate the metabolic similarities and differences, both species were treated with pharmacological inhibitors, confirming a lack of essential electron transport chain activity inT. congolense,but increased sensitivity to inhibition of mitochondrial pyruvate import. Strikingly,T. congolenseexhibited significant resistance to inhibitors of fatty acid synthesis, including a 780-fold greater EC50against the lipase and fatty acid synthase inhibitor Orlistat, compared toT. brucei. These data highlight that bloodstream formT. congolensediverges fromT. bruceiin key areas of metabolism, with several features that are intermediate between bloodstream- and insect-stageT. brucei. These results have implications for drug development, mechanisms of drug resistance and host-pathogen interactions.

Significance Parasites are interesting models for studying speciation processes because they have a high potential for specialization, thanks to the intimate ecological association with their hosts and vectors. Yet little is known about the circumstances under which new parasite lineages emerge. Here we studied the genome diversity of parasites of the Leishmania braziliensis species complex that inhabit both Amazonian and Andean biotas in Peru. We identify three major parasite lineages that occupy particular ecological niches and show that these emerged during forestation changes over the past 150,000 y. We furthermore discovered that meiotic recombination between Amazonian and Andean lineages resulted in full-genome hybrids presenting mixed mitochondrial genomes, providing insights into the genetic consequences of hybridization in parasitic protozoa., The tropical Andes are an important natural laboratory to understand speciation in many taxa. Here we examined the evolutionary history of parasites of the Leishmania braziliensis species complex based on whole-genome sequencing of 67 isolates from 47 localities in Peru. We first show the origin of Andean Leishmania as a clade of near-clonal lineages that diverged from admixed Amazonian ancestors, accompanied by a significant reduction in genome diversity and large structural variations implicated in host–parasite interactions. Within the Andean species, patterns of population structure were strongly associated with biogeographical origin. Molecular clock and ecological niche modeling suggested that the history of diversification of the Andean lineages is limited to the Late Pleistocene and intimately associated with habitat contractions driven by climate change. These results suggest that changes in forestation over the past 150,000 y have influenced speciation and diversity of these Neotropical parasites. Second, genome-scale analyses provided evidence of meiotic-like recombination between Andean and Amazonian Leishmania species, resulting in full-genome hybrids. The mitochondrial genome of these hybrids consisted of homogeneous uniparental maxicircles, but minicircles originated from both parental species. We further show that mitochondrial minicircles—but not maxicircles—show a similar evolutionary pattern to the nuclear genome, suggesting that compatibility between nuclear-encoded mitochondrial genes and minicircle-encoded guide RNA genes is essential to maintain efficient respiration. By comparing full nuclear and mitochondrial genome ancestries, our data expand our appreciation on the genetic consequences of diversification and hybridization in parasitic protozoa.

Trypanosome U-insertion/deletion RNA editing in mitochondrial mRNAs involves guide RNAs (gRNAs) and the auxiliary RNA editing substrate binding complex (RESC) and RNA editing helicase 2 complex (REH2C). RESC and REH2C stably copurify with editing mRNAs but the functional interplay between these complexes remains unclear. Most steady-state mRNAs are partially edited and include misedited “junction” regions that match neither pre-mRNA nor fully edited transcripts. Editing specificity is central to mitochondrial RNA maturation and function, but its basic control mechanisms remain unclear. Here we applied a novel nucleotide-resolution RNA-seq approach to examine ribosomal protein subunit 12 (RPS12) and ATPase subunit 6 (A6) mRNA transcripts. We directly compared transcripts associated with RESC and REH2C to those found in total mitochondrial RNA. RESC-associated transcripts exhibited site-preferential enrichments in total and accurate edits. REH2C loss-of-function induced similar substrate-specific and site-specific editing effects in total and RESC-associated RNA. It decreased total editing primarily at RPS12 5′ positions but increased total editing at examined A6 3′ positions. REH2C loss-of-function caused site-preferential loss of accurate editing in both transcripts. However, changes in total or accurate edits did not necessarily involve common sites. A few 5′ nucleotides of the initiating gRNA (gRNA-1) directed accurate editing in both transcripts. However, in RPS12, two conserved 3′-terminal adenines in gRNA-1 could direct a noncanonical 2U-insertion that causes major pausing in 3′–5′ progression. In A6, a noncanonical sequence element that depends on REH2C in a region normally targeted by the 3′ half of gRNA-1 may hinder early editing progression. Overall, we defined transcript-specific effects of REH2C loss.

Mitochondrial ATP synthase is a reversible nanomotor synthesizing or hydrolyzing ATP depending on the potential across the membrane in which it is embedded. In the unicellular parasite Trypanosoma brucei, the direction of the complex depends on the life cycle stage of this digenetic parasite: in the midgut of the tsetse fly vector (procyclic form), the FoF1–ATP synthase generates ATP by oxidative phosphorylation, whereas in the mammalian bloodstream form, this complex hydrolyzes ATP and maintains mitochondrial membrane potential (ΔΨm). The trypanosome FoF1–ATP synthase contains numerous lineage-specific subunits whose roles remain unknown. Here, we seek to elucidate the function of the lineage-specific protein Tb1, the largest membrane-bound subunit. In procyclic form cells, Tb1 silencing resulted in a decrease of FoF1–ATP synthase monomers and dimers, rerouting of mitochondrial electron transfer to the alternative oxidase, reduced growth rate and cellular ATP levels, and elevated ΔΨm and total cellular reactive oxygen species levels. In bloodstream form parasites, RNAi silencing of Tb1 by ∼90% resulted in decreased FoF1–ATPase monomers and dimers, but it had no apparent effect on growth. The same findings were obtained by silencing of the oligomycin sensitivity-conferring protein, a conserved subunit in T. brucei FoF1–ATP synthase. However, as expected, nearly complete Tb1 or oligomycin sensitivity-conferring protein suppression was lethal because of the inability to sustain ΔΨm. The diminishment of FoF1–ATPase complexes was further accompanied by a decreased ADP/ATP ratio and reduced oxygen consumption via the alternative oxidase. Our data illuminate the often diametrically opposed bioenergetic consequences of FoF1–ATP synthase loss in insect versus mammalian forms of the parasite.

Single mRNA molecules are frequently detected by single molecule fluorescence in situ hybridisation (smFISH) using branched DNA technology. While providing strong and background-reduced signals, the method is inefficient in detecting mRNAs within dense structures, in monitoring mRNA compactness and in quantifying abundant mRNAs.To overcome these limitations, we have hybridised slices of high pressure frozen, LR White embedded cells (LR White smFISH). mRNA detection is physically restricted to the surface of the resin. This enables single molecule detection of RNAs with accuracy comparable to RNA sequencing, irrespective of their abundance, while at the same time providing spatial information on RNA localisation that can be complemented with immunofluorescence and electron microscopy, as well as electron tomography. Moreover, LR White embedding restricts the number of available probe pair recognition sites for each mRNA to a small subset. As a consequence, differences in signal intensities between RNA populations reflect differences in RNA tertiary structures, and we show that the method can be employed to probe for mRNA compactness. We apply LR White smFISH to answer some outstanding questions related to trans-splicing, RNA granules and mitochondrial RNA editing, using trypanosomes and their versatile RNA biology as a model system.

Trypanosoma brucei spp. cause African human and animal trypanosomiasis, a burden on health and economy in Africa. These hemoflagellates are distinguished by a kinetoplast nucleoid containing mitochondrial DNAs of two kinds: maxicircles encoding ribosomal RNAs (rRNAs) and proteins and minicircles bearing guide RNAs (gRNAs) for mRNA editing. All RNAs are produced by a phage-type RNA polymerase as 3′ extended precursors, which undergo exonucleolytic trimming. Most pre-mRNAs proceed through 3′ adenylation, uridine insertion/deletion editing, and 3′ A/U-tailing. The rRNAs and gRNAs are 3′ uridylated. Historically, RNA editing has attracted major research effort, and recently essential pre- and postediting processing events have been discovered. Here, we classify the key players that transform primary transcripts into mature molecules and regulate their function and turnover.

Ribosome biosynthesis, best studied in opisthokonts, is a highly complex process involving numerous protein and RNA factors. Yet, very little is known about the early stages of pre-18S rRNA processing even in these model organisms, let alone the conservation of this mechanism in other eukaryotes. Here we extend our knowledge of this process by identifying and characterizing the essential protein TbUTP10, a homolog of yeast U3 small nucleolar RNA-associated protein 10 - UTP10 (HEATR1 in human), in the excavate parasitic protist Trypanosoma brucei. We show that TbUTP10 localizes to the nucleolus and that its ablation by RNAi knock-down in two different T. brucei life cycle stages results in similar phenotypes: a disruption of pre-18S rRNA processing, exemplified by the accumulation of rRNA precursors, a reduction of mature 18S rRNA, and also a decrease in the level of U3 snoRNA. Moreover, polysome profiles of the RNAi-induced knock-down cells show a complete disappearance of the 40S ribosomal subunit, and a prominent accumulation of the 60S large ribosomal subunit, reflecting impaired ribosome assembly. Thus, TbUTP10 is an important protein in the processing of 18S rRNA.

Mitochondrial ATP synthase is a reversible nanomotor synthesizing or hydrolyzing ATP depending on the potential across the membrane in which it is embedded. In the unicellular parasite Trypanosoma brucei, the direction of the complex depends on the life cycle stage of this digenetic parasite: in the midgut of the tsetse fly vector (procyclic form), the FoF1–ATP synthase generates ATP by oxidative phosphorylation, whereas in the mammalian bloodstream form, this complex hydrolyzes ATP and maintains mitochondrial membrane potential (ΔΨm). The trypanosome FoF1–ATP synthase contains numerous lineage-specific subunits whose roles remain unknown. Here, we seek to elucidate the function of the lineage-specific protein Tb1, the largest membrane-bound subunit. In procyclic form cells, Tb1 silencing resulted in a decrease of FoF1–ATP synthase monomers and dimers, rerouting of mitochondrial electron transfer to the alternative oxidase, reduced growth rate and cellular ATP levels, and elevated ΔΨm and total cellular reactive oxygen species levels. In bloodstream form parasites, RNAi silencing of Tb1 by ∼90% resulted in decreased FoF1–ATPase monomers and dimers, but it had no apparent effect on growth. The same findings were obtained by silencing of the oligomycin sensitivity-conferring protein, a conserved subunit in T. brucei FoF1–ATP synthase. However, as expected, nearly complete Tb1 or oligomycin sensitivity-conferring protein suppression was lethal because of the inability to sustain ΔΨm. The diminishment of FoF1–ATPase complexes was further accompanied by a decreased ADP/ATP ratio and reduced oxygen consumption via the alternative oxidase. Our data illuminate the often diametrically opposed bioenergetic consequences of FoF1–ATP synthase loss in insect versus mammalian forms of the parasite.

Trypanosomatid parasites cause diseases in humans and livestock. It was reported that partial inhibition of the vacuolar ATPase (V-ATPase) affects the dependence of

We used electron cryotomography and subtomogram averaging to determine the in situ structures of mitochondrial ATP synthase dimers from two organisms belonging to the phylum euglenozoa: Trypanosoma brucei, a lethal human parasite, and Euglena gracilis, a photosynthetic protist. At a resolution of 32.5 Å and 27.5 Å, respectively, the two structures clearly exhibit a noncanonical F1 head, in which the catalytic (αβ)3 assembly forms a triangular pyramid rather than the pseudo-sixfold ring arrangement typical of all other ATP synthases investigated so far. Fitting of known X-ray structures reveals that this unusual geometry results from a phylum-specific cleavage of the α subunit, in which the C-terminal αC fragments are displaced by ∼20 Å and rotated by ∼30° from their expected positions. In this location, the αC fragment is unable to form the conserved catalytic interface that was thought to be essential for ATP synthesis, and cannot convert γ-subunit rotation into the conformational changes implicit in rotary catalysis. The new arrangement of catalytic subunits suggests that the mechanism of ATP generation by rotary ATPases is less strictly conserved than has been generally assumed. The ATP synthases of these organisms present a unique model system for discerning the individual contributions of the α and β subunits to the fundamental process of ATP synthesis.

Mitochondria cannot form de novo but require mechanisms allowing their inheritance to daughter cells. In contrast to most other eukaryotes Trypanosoma brucei has a single mitochondrion whose single-unit genome is physically connected to the flagellum. Here we identify a β-barrel mitochondrial outer membrane protein, termed tripartite attachment complex 40 (TAC40), that localizes to this connection. TAC40 is essential for mitochondrial DNA inheritance and belongs to the mitochondrial porin protein family. However, it is not specifically related to any of the three subclasses of mitochondrial porins represented by the metabolite transporter voltage-dependent anion channel (VDAC), the protein translocator of the outer membrane 40 (TOM40), or the fungi-specific MDM10, a component of the endoplasmic reticulum-mitochondria encounter structure (ERMES). MDM10 and TAC40 mediate cellular architecture and participate in transmembrane complexes that are essential for mitochondrial DNA inheritance. In yeast MDM10, in the context of the ERMES, is postulated to connect the mitochondrial genomes to actin filaments, whereas in trypanosomes TAC40 mediates the linkage of the mitochondrial DNA to the basal body of the flagellum. However, TAC40 does not colocalize with trypanosomal orthologs of ERMES components and, unlike MDM10, it regulates neither mitochondrial morphology nor the assembly of the protein translocase. TAC40 therefore defines a novel subclass of mitochondrial porins that is distinct from VDAC, TOM40, and MDM10. However, whereas the architecture of the TAC40-containing complex in trypanosomes and the MDM10-containing ERMES in yeast is very different, both are organized around a β-barrel protein of the mitochondrial porin family that mediates a DNA-cytoskeleton linkage that is essential for mitochondrial DNA inheritance.

One of the most promising new targets for trypanocidal drugs to emerge in recent years is the cyclic AMP (cAMP) phosphodiesterase (PDE) activity encoded by TbrPDEB1 and TbrPDEB2 . These genes were genetically confirmed as essential, and a high-affinity inhibitor, CpdA, displays potent antitrypanosomal activity. To identify effectors of the elevated cAMP levels resulting from CpdA action and, consequently, potential sites for adaptations giving resistance to PDE inhibitors, resistance to the drug was induced. Selection of mutagenized trypanosomes resulted in resistance to CpdA as well as cross-resistance to membrane-permeable cAMP analogues but not to currently used trypanocidal drugs. Resistance was not due to changes in cAMP levels or in PDEB genes. A second approach, a genome-wide RNA interference (RNAi) library screen, returned four genes giving resistance to CpdA upon knockdown. Validation by independent RNAi strategies confirmed resistance to CpdA and suggested a role for the identified cA MP R esponse P roteins (CARPs) in cAMP action. CARP1 is unique to kinetoplastid parasites and has predicted cyclic nucleotide binding-like domains, and RNAi repression resulted in >100-fold resistance. CARP2 and CARP4 are hypothetical conserved proteins associated with the eukaryotic flagellar proteome or with flagellar function, with an orthologue of CARP4 implicated in human disease. CARP3 is a hypothetical protein, unique to Trypanosoma . CARP1 to CARP4 likely represent components of a novel cAMP signaling pathway in the parasite. As cAMP metabolism is validated as a drug target in Trypanosoma brucei , cAMP effectors highly divergent from the mammalian host, such as CARP1 , lend themselves to further pharmacological development.

African trypanosomes, except Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense, which cause human African trypanosomiasis, are lysed by the human serum protein apolipoprotein L1 (ApoL1). These two subspecies can resist human ApoL1 because they express the serum resistance proteins T. b. gambiense glycoprotein (TgsGP) and serum resistance-associated protein (SRA), respectively. Whereas in T. b. rhodesiense, SRA is necessary and sufficient to inhibit ApoL1, in T. b. gambiense, TgsGP cannot protect against high ApoL1 uptake, so different additional mechanisms contribute to limit this uptake. Here we report a complex interplay between trypanosomes and an ApoL1 variant, revealing important insights into innate human immunity against these parasites. Using whole-genome sequencing, we characterized an atypical T. b. gambiense infection in a patient in Ghana. We show that the infecting trypanosome has diverged from the classical T. b. gambiense strains and lacks the TgsGP defense mechanism against human serum. By sequencing the ApoL1 gene of the patient and subsequent in vitro mutagenesis experiments, we demonstrate that a homozygous missense substitution (N264K) in the membrane-addressing domain of this ApoL1 variant knocks down the trypanolytic activity, allowing the trypanosome to avoid ApoL1-mediated immunity., SCOPUS: ar.j, info:eu-repo/semantics/published

The structure of a C11 peptidase PmC11 from the gut bacterium, Parabacteroides merdae, has recently been determined, enabling the identification and characterization of a C11 orthologue, PNT1, in the parasitic protozoon Trypanosoma brucei. A phylogenetic analysis identified PmC11 orthologues in bacteria, archaea, Chromerids, Coccidia, and Kinetoplastida, the latter being the most divergent. A primary sequence alignment of PNT1 with clostripain and PmC11 revealed the position of the characteristic His-Cys catalytic dyad (His(99) and Cys(136)), and an Asp (Asp(134)) in the potential S1 binding site. Immunofluorescence and cryoelectron microscopy revealed that PNT1 localizes to the kinetoplast, an organelle containing the mitochondrial genome of the parasite (kDNA), with an accumulation of the protein at or near the antipodal sites. Depletion of PNT1 by RNAi in the T. brucei bloodstream form was lethal both in in vitro culture and in vivo in mice and the induced population accumulated cells lacking a kinetoplast. In contrast, overexpression of PNT1 led to cells having mislocated kinetoplasts. RNAi depletion of PNT1 in a kDNA independent cell line resulted in kinetoplast loss but was viable, indicating that PNT1 is required exclusively for kinetoplast maintenance. Expression of a recoded wild-type PNT1 allele, but not of an active site mutant restored parasite viability after induction in vitro and in vivo confirming that the peptidase activity of PNT1 is essential for parasite survival. These data provide evidence that PNT1 is a cysteine peptidase that is required exclusively for maintenance of the trypanosome kinetoplast.

Trypanosomes show an intriguing organization of their mitochondrial DNA into a catenated network, the kinetoplast DNA (kDNA). While more than 30 proteins involved in kDNA replication have been described, only few components of kDNA segregation machinery are currently known. Electron microscopy studies identified a high-order structure, the tripartite attachment complex (TAC), linking the basal body of the flagellum via the mitochondrial membranes to the kDNA. Here we describe TAC102, a novel core component of the TAC, which is essential for proper kDNA segregation during cell division. Loss of TAC102 leads to mitochondrial genome missegregation but has no impact on proper organelle biogenesis and segregation. The protein is present throughout the cell cycle and is assembled into the newly developing TAC only after the pro-basal body has matured indicating a hierarchy in the assembly process. Furthermore, we provide evidence that the TAC is replicated de novo rather than using a semi-conservative mechanism. Lastly, we demonstrate that TAC102 lacks an N-terminal mitochondrial targeting sequence and requires sequences in the C-terminal part of the protein for its proper localization., Author Summary Proper segregation of the mitochondrial genome during cell division is a prerequisite of healthy eukaryotic cells. However, the mechanism underlying the segregation process is only poorly understood. We use the single celled parasite Trypanosoma brucei, which, unlike most model organisms, harbors a single large mitochondrion with a single mitochondrial genome, also called kinetoplast DNA (kDNA), to study this question. In trypanosomes, kDNA replication and segregation are tightly integrated into the cell cycle and thus can be studied alongside cell cycle markers. Furthermore, previous studies using electron microscopy have characterized the tripartite attachment complex (TAC) as a structural element of the mitochondrial genome segregation machinery. Here, we characterize TAC102, a novel trypanosome protein localized to the TAC. The protein is essential for proper kDNA segregation and cell growth. We analyze the presence of this protein using super resolution microscopy and show that TAC102 is a mitochondrial protein localized between the kDNA and the basal body of the cell’s flagellum. In addition, we characterize different parts of the protein and show that the C-terminus of TAC102 is important for its proper localization. The data and resources presented will allow a more detailed characterization of the dynamics and hierarchy of the TAC in the future and might open new avenues for drug discovery targeting this structure.

Two key biological features distinguish Trypanosoma evansi from the T. brucei group: independence from the tsetse fly as obligatory vector, and independence from the need for functional mitochondrial DNA (kinetoplast or kDNA). In an effort to better understand the molecular causes and consequences of these differences, we sequenced the genome of an akinetoplastic T. evansi strain from China and compared it to the T. b. brucei reference strain. The annotated T. evansi genome shows extensive similarity to the reference, with 94.9% of the predicted T. b. brucei coding sequences (CDS) having an ortholog in T. evansi, and 94.6% of the non-repetitive orthologs having a nucleotide identity of 95% or greater. Interestingly, several procyclin-associated genes (PAGs) were disrupted or not found in this T. evansi strain, suggesting a selective loss of function in the absence of the insect life-cycle stage. Surprisingly, orthologous sequences were found in T. evansi for all 978 nuclear CDS predicted to represent the mitochondrial proteome in T. brucei, although a small number of these may have lost functionality. Consistent with previous results, the F1FO-ATP synthase γ subunit was found to have an A281 deletion, which is involved in generation of a mitochondrial membrane potential in the absence of kDNA. Candidates for CDS that are absent from the reference genome were identified in supplementary de novo assemblies of T. evansi reads. Phylogenetic analyses show that the sequenced strain belongs to a dominant group of clonal T. evansi strains with worldwide distribution that also includes isolates classified as T. equiperdum. At least three other types of T. evansi or T. equiperdum have emerged independently. Overall, the elucidation of the T. evansi genome sequence reveals extensive similarity of T. brucei and supports the contention that T. evansi should be classified as a subspecies of T. brucei., Author Summary The single-cell parasite Trypanosoma evansi is the disease-causing trypanosome with the widest geographical distribution. The disease, called surra, has significant economic impact primarily due to infections of cattle, horses, and camels. Morphologically the parasite is indistinguishable from bloodstream stage T. brucei, a parasite causing sleeping sickness in humans and the disease nagana in animals. T. brucei, however, is strictly bound to sub-Saharan Africa where its obligate vector, the tsetse fly, resides. The lack of a complete mitochondrial genome in T. evansi further distinguishes this parasite from T. brucei. Important questions regarding the biology of T. evansi include how it escaped from Africa, whether this has happened more than once, and how exactly it is related to T. brucei. To help answer these questions we have sequenced the T. evansi nuclear genome. Our phylogenetic analysis demonstrates that T. evansi, and the closely related horse parasite T. equiperdum, evolved more than once from T. brucei. We also demonstrate extensive similarity to T. brucei, including the maintenance of numerous genes that T. evansi no longer requires. Therefore, despite the significant functional and pathological differences between T. evansi and T. brucei, our analysis supports the notion that T. evansi is not an independent species.

Survival of bloodstream form Trypanosoma brucei, the agent of African sleeping sickness, normally requires mitochondrial gene expression, despite the absence of oxidative phosphorylation in this stage of the parasite's life cycle. Here we report that silencing expression of the α subunit of the mitochondrial F1-ATP synthase complex is lethal for bloodstream stage T. brucei as well as for T. evansi, a closely related species that lacks mitochondrial protein coding genes (i.e. is dyskinetoplastic). Our results suggest that the lethal effect is due to collapse of the mitochondrial membrane potential, which is required for mitochondrial function and biogenesis. We also identified a mutation in the γ subunit of F1 that is likely to be involved in circumventing the requirement for mitochondrial gene expression in another dyskinetoplastic form. Our data reveal that the mitochondrial ATP synthase complex functions in the bloodstream stage opposite to that in the insect stage and in most other eukaryotes, namely using ATP hydrolysis to generate the mitochondrial membrane potential.

The cDNAs for two isoforms (I and II) of the 14-3-3 proteins have been cloned and functionally characterized in Trypanosoma brucei. The amino acid sequences of isoforms I and II have 47 and 50% identity to the human tau isoform, respectively, with important conserved features including a potential amphipathic groove for the binding of phosphoserine/phosphothreonine-containing motifs and a nuclear export signal-like domain. Both isoforms are abundantly expressed at approximately equal levels (1-2 x 10(6) molecules/cell) and localized mainly in the cytoplasm. Knockdown by induction of double-stranded RNA of isoform I and/or II in both bloodstream and procyclic forms resulted first in a reduction of cell motility and then significant reduction in cell growth rates and morphological changes; the changes include aberrant numbers of organelles and abnormal shapes and sizes that mimic phenotypes produced by various cytokinesis inhibitors. Morphological and fluorescence-activated cell sorting analysis of the cell cycle suggested that isoforms I and II might play important roles in nuclear (G2-M transition) and cell (M-G1 transition) division. These findings indicate that the 14-3-3 proteins play important roles in cell motility, cytokinesis, and the cell cycle.

Most mitochondrial mRNAs in kinetoplastids require editing, that is, the posttranscriptional insertion and deletion of uridine nucleotides that are specified by guide RNAs and catalyzed by multiprotein complexes. Recent studies have identified many of the proteins in these complexes, in addition to some of their functions and interactions. Although much remains unknown, a picture of highly organized complexes is emerging that shows that the complex that catalyzes the central steps of editing is partitioned into distinct insertion and deletion editing subcomplexes. These subcomplexes coordinate hundreds of ordered catalytic steps that function to produce a single mature mRNA. The dynamic processes, which might entail interactions among multiprotein complexes and changes in their composition and conformation, remain to be elucidated.

Trypanosomatids are causative agents of several devastating tropical diseases such as African sleeping sickness, Chagas’ disease and leishmaniasis. There are no effective vaccines available to date for treatment of these protozoan diseases, while current drugs have limited efficacy, significant toxicity and suffer from increasing resistance. Trypanosomatids have several remarkable and unique metabolic and structural features that are of great interest for developing new anti-protozoan therapeutics. One such feature is “RNA editing”, an essential process in these pathogenic protozoa. Transcripts for key trypanosomatid mitochondrial proteins undergo extensive post-transcriptional RNA editing by specifically inserting or deleting uridylates from premature mRNA in order to create mature mRNAs that encode functional proteins. The RNA editing process is carried out in aw1.6 MDa multi-protein complex, the editosome. In Trypanosoma brucei, one of the editosome’s core enzymes, the RNA editing ligase 1 (TbREL1), has been shown to be essential for survival of both insect and bloodstream forms of the parasite. We report here the crystal structure of the catalytic domain of TbREL1 at 1.2 A u resolution, in complex with ATP and magnesium. The magnesium ion interacts with the b and g-phosphate groups and is almost perfectly octahedrally coordinated by six phosphate and water oxygen atoms. ATP makes extensive direct and indirect interactions with the ligase via essentially all its atoms while extending its base into a deep pocket. In addition, the ATP makes numerous interactions with residues that are conserved in the editing ligases only. Further away from the active site, TbREL1 contains a unique loop containing several hydrophobic residues that are highly conserved among trypanosomatid RNA editing ligases which may play a role in protein– protein interactions in the editosome. The distinct characteristics of the adenine-binding pocket, and the absence of any close homolog in the human genome, bode well for the design of selective inhibitors that will block the essential RNA ligase function in a number of major protozoan pathogens. q 2004 Elsevier Ltd. All rights reserved.

The Trypanosoma brucei editosome catalyzes the maturation of mitochondrial mRNAs through the insertion and deletion of uridylates and contains at least 16 stably associated proteins. We examined physical and functional associations among these proteins using three different approaches: purification of complexes via tagged editing ligases TbREL1 and TbREL2, comprehensive yeast two-hybrid analysis, and coimmunoprecipitation of recombinant proteins. A purified TbREL1 subcomplex catalyzed precleaved deletion editing in vitro, while a purified TbREL2 subcomplex catalyzed precleaved insertion editing in vitro. The TbREL1 subcomplex contained three to four proteins, including a putative exonuclease, and appeared to be coordinated by the zinc finger protein TbMP63. The TbREL2 subcomplex had a different composition, contained the TbMP57 terminal uridylyl transferase, and appeared to be coordinated by the TbMP81 zinc finger protein. This study provides insight into the molecular architecture of the editosome and supports the existence of separate subcomplexes for deletion and insertion editing.

RNA editing in trypanosomes occurs by a series of enzymatic steps that are catalyzed by a macromolecular complex. The TbMP52 protein is shown to be a component of this complex, to have RNA ligase activity, and to be one of two adenylatable proteins in the complex. Regulated repression ofTbMP52blocks editing, which shows that it is a functional component of the editing complex. This repression is lethal in bloodforms of the parasite, indicating that editing is essential in the mammalian stage of the life cycle. The editing complex, which is present in all kinetoplastid parasites, may thus be a chemotherapeutic target.