KREH2 helicase represses ND7 mRNA editing in procyclic-stage Trypanosoma brucei by opposite modulation of canonical and 'moonlighting' gRNA utilization creating a proposed mRNA structure.

Unknown factors regulate mitochondrial U-insertion/deletion (U-indel) RNA editing in procyclic-form (PCF) and bloodstream-form (BSF) T. brucei. This editing, directed by anti-sense gRNAs, creates canonical protein-encoding mRNAs and may developmentally control respiration.

Trypanosome RNA helicase KREH2 differentially controls non-canonical editing and putative repressive structure via a novel proposed 'bifunctional' gRNA in mRNA A6.

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.

Metabolic insights into phosphofructokinase inhibition in bloodstream-form trypanosomes.

Previously, we reported the development of novel small molecules that are potent inhibitors of the glycolytic enzyme phosphofructokinase (PFK) of and related protists responsible for serious diseases in humans and domestic animals. Cultured bloodstream-form trypanosomes, which are fully reliant on glycolysis for their ATP production, are rapidly killed at submicromolar concentrations of these compounds, which have no effect on the activity of human PFKs and human cells. Single-day oral dosing cures stage 1 human trypanosomiasis in an animal model.

Deep kinetoplast genome analyses result in a novel molecular assay for detecting -specific minicircles.

The World Health Organization targeted () human African trypanosomiasis for elimination of transmission by 2030. Sensitive molecular markers that specifically detect type 1 () parasites will be important tools to assist in reaching this goal. We aim at improving molecular diagnosis of 1 infections by targeting the abundant mitochondrial minicircles within the kinetoplast of these parasites.

Molecular Analysis of Trypanosome Infections in Algerian Camels.

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.

Organization of minicircle cassettes and guide RNA genes in .

Mitochondrial DNA of protists of order 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.

Oxidative Phosphorylation Is Required for Powering Motility and Development of the Sleeping Sickness Parasite Trypanosoma brucei in the Tsetse Fly Vector.

The single-celled parasite Trypanosoma brucei is transmitted by hematophagous tsetse flies. Life cycle 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 colonizes the glucose-poor insect midgut, ATP production is thought to depend on activation of mitochondrial amino acid catabolism via oxidative phosphorylation (OXPHOS).

A Novel High-Content Phenotypic Screen To Identify Inhibitors of Mitochondrial DNA Maintenance in Trypanosomes.

Kinetoplastid parasites cause diverse neglected diseases in humans and livestock, with an urgent need for new treatments. The survival of kinetoplastids depends on their uniquely structured mitochondrial genome (kDNA), the eponymous kinetoplast. Here, we report the development of a high-content screen for pharmacologically induced kDNA loss, based on specific staining of parasites and automated image analysis.

rKOMICS: an R package for processing mitochondrial minicircle assemblies in population-scale genome projects.

Background: The advent of population-scale genome projects has revolutionized our biological understanding of parasitic protozoa. However, while hundreds to thousands of nuclear genomes of parasitic protozoa have been generated and analyzed, information about the diversity, structure and evolution of their mitochondrial genomes remains fragmentary, mainly because of their extraordinary complexity. Indeed, unicellular flagellates of the order Kinetoplastida contain structurally the most complex mitochondrial genome of all eukaryotes, organized as a giant network of homogeneous maxicircles and heterogeneous minicircles.

Parallel monitoring of RNA abundance, localization and compactness with correlative single molecule FISH on LR White embedded samples.

Single mRNA molecules are frequently detected by single molecule fluorescence in situ hybridization (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 hybridized slices of high pressure frozen, freeze-substituted and LR White embedded cells (LR White smFISH).

Ecological divergence and hybridization of Neotropical parasites.

The tropical Andes are an important natural laboratory to understand speciation in many taxa. Here we examined the evolutionary history of parasites of the species complex based on whole-genome sequencing of 67 isolates from 47 localities in Peru. We first show the origin of Andean 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.

Site-specific and substrate-specific control of accurate mRNA editing by a helicase complex in trypanosomes.

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.

Assembly and annotation of the mitochondrial minicircle genome of a differentiation-competent strain of Trypanosoma brucei.

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.

Emerging challenges in understanding trypanosome antigenic variation.

Many pathogens evade host immunity by periodically changing the proteins they express on their surface - a phenomenon termed antigenic variation. An extreme form of antigenic variation, based around switching the composition of a Variant Surface Glycoprotein (VSG) coat, is exhibited by the African trypanosome , which causes human disease. The molecular details of VSG switching in have been extensively studied over the last three decades, revealing in increasing detail the machinery and mechanisms by which VSG expression is controlled and altered.

TbUTP10, a protein involved in early stages of pre-18S rRNA processing in Trypanosoma brucei.

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.

Pharmacological Inhibition of the Vacuolar ATPase in Bloodstream-Form Trypanosoma brucei Rescues Genetic Knockdown of Mitochondrial Gene Expression.

Trypanosomatid parasites cause diseases in humans and livestock. It was reported that partial inhibition of the vacuolar ATPase (V-ATPase) affects the dependence of on its mitochondrial genome (kinetoplast DNA [kDNA]), a target of the antitrypanosomatid drug isometamidium. Here, we report that V-ATPase inhibition with bafilomycin A1 (BafA) provides partial resistance to genetic knockdown of mitochondrial gene expression.

Multiple evolutionary origins of Trypanosoma evansi in Kenya.

Trypanosoma evansi is the parasite causing surra, a form of trypanosomiasis in camels and other livestock, and a serious economic burden in Kenya and many other parts of the world. Trypanosoma evansi transmission can be sustained mechanically by tabanid and Stomoxys biting flies, whereas the closely related African trypanosomes T. brucei brucei and T.

In situ structure of trypanosomal ATP synthase dimer reveals a unique arrangement of catalytic subunits.

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.

NADH dehydrogenase of Trypanosoma brucei is important for efficient acetate production in bloodstream forms.

In the slender bloodstream form, Trypanosoma brucei mitochondria are repressed for many functions. Multiple components of mitochondrial complex I, NADH:ubiquinone oxidoreductase, are expressed in this stage, but electron transfer through complex I is not essential. Here we investigate the role of the parasite's second NADH:ubiquinone oxidoreductase, NDH2, which is composed of a single subunit that also localizes to the mitochondrion.

Reduced Mitochondrial Membrane Potential Is a Late Adaptation of Trypanosoma brucei brucei to Isometamidium Preceded by Mutations in the γ Subunit of the F1Fo-ATPase.

Background: Isometamidium is the main prophylactic drug used to prevent the infection of livestock with trypanosomes that cause Animal African Trypanosomiasis. As well as the animal infective trypanosome species, livestock can also harbor the closely related human infective subspecies T. b.