Identification of novel components of Trypanosoma brucei editosomes

The editosome is a multiprotein complex that catalyzes the insertion and deletion of uridylates that occurs during RNA editing in trypanosomatids. We report the identification of nine novel editosome proteins in Trypanosoma brucei. They were identified by mass spectrometric analysis of functional editosomes that were purified by serial ion exchange/gel permeation chromatography, immunoaffinity chromatography specific to the TbMP63 editosome protein, or tandem affinity purification based on a tagged RNA editing ligase. The newly identified proteins have ribonuclease and/or RNA binding motifs suggesting nuclease function for at least some of these. Five of the proteins are interrelated, as are two others, and one is related to four previously identified editosome proteins. The implications of these findings are discussed

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). This process involves respiratory chain complexes and F1Fo-ATP synthase and requires protein subunits of these complexes that are encoded in the parasite's mitochondrial DNA (kDNA). Here, we show that progressive loss of kDNA-encoded functions correlates with a decreasing ability to initiate and complete development in the tsetse. First, parasites with a mutated F1Fo-ATP synthase with reduced capacity for OXPHOS can initiate differentiation from bloodstream to insect form, but they are unable to proliferate in vitro. Unexpectedly, these cells can still colonize the tsetse midgut. However, these parasites exhibit a motility defect and are severely impaired in colonizing or migrating to subsequent tsetse tissues. Second, parasites with a fully disrupted F1Fo-ATP synthase complex that is completely unable to produce ATP by OXPHOS 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, parasites lacking kDNA entirely can initiate differentiation but die soon after. Together, these scenarios suggest that efficient ATP production via OXPHOS is not essential for initial colonization of the tsetse vector but is required to power trypanosome migration within the fly. IMPORTANCE African trypanosomes cause disease in humans and their livestock and are transmitted by tsetse flies. The insect ingests these parasites with its blood meal, but to be transmitted to another mammal, the trypanosome must undergo complex development within the tsetse fly and migrate from the insect's gut to its salivary glands. Crucially, the parasite must switch from a sugar-based diet while in the mammal to a diet based primarily on amino acids when it develops in the insect. Here, we show that efficient energy production by an organelle called the mitochondrion is critical for the trypanosome's ability to swim and to migrate through the tsetse fly. Surprisingly, trypanosomes with impaired mitochondrial energy production are only mildly compromised in their ability to colonize the tsetse fly midgut. Our study adds a new perspective to the emerging view that infection of tsetse flies by trypanosomes is more complex than previously thought

Functional and Structural Insights Revealed by Molecular Dynamics Simulations of an Essential RNA Editing Ligase in Trypanosoma brucei

RNA editing ligase 1 (TbREL1) is required for the survival of both the insect and bloodstream forms of Trypanosoma brucei, the parasite responsible for the devastating tropical disease African sleeping sickness. The type of RNA editing that TbREL1 is involved in is unique to the trypanosomes, and no close human homolog is known to exist. In addition, the high-resolution crystal structure revealed several unique features of the active site, making this enzyme a promising target for structure-based drug design. In this work, two 20 ns atomistic molecular dynamics (MD) simulations are employed to investigate the dynamics of TbREL1, both with and without the ATP substrate present. The flexibility of the active site, dynamics of conserved residues and crystallized water molecules, and the interactions between TbREL1 and the ATP substrate are investigated and discussed in the context of TbREL1's function. Differences in local and global motion upon ATP binding suggest that two peripheral loops, unique to the trypanosomes, may be involved in interdomain signaling events. Notably, a significant structural rearrangement of the enzyme's active site occurs during the apo simulations, opening an additional cavity adjacent to the ATP binding site that could be exploited in the development of effective inhibitors directed against this protozoan parasite. Finally, ensemble averaged electrostatics calculations over the MD simulations reveal a novel putative RNA binding site, a discovery that has previously eluded scientists. Ultimately, we use the insights gained through the MD simulations to make several predictions and recommendations, which we anticipate will help direct future experimental studies and structure-based drug discovery efforts against this vital enzyme

The F0F1-ATP Synthase Complex Contains Novel Subunits and Is Essential for Procyclic Trypanosoma brucei

The mitochondrial F0F1 ATP synthase is an essential multi-subunit protein complex in the vast majority of eukaryotes but little is known about its composition and role in Trypanosoma brucei, an early diverged eukaryotic pathogen. We purified the F0F1 ATP synthase by a combination of affinity purification, immunoprecipitation and blue-native gel electrophoresis and characterized its composition and function. We identified 22 proteins of which five are related to F1 subunits, three to F0 subunits, and 14 which have no obvious homology to proteins outside the kinetoplastids. RNAi silencing of expression of the F1 α subunit or either of the two novel proteins showed that they are each essential for the viability of procyclic (insect stage) cells and are important for the structural integrity of the F0F1-ATP synthase complex. We also observed a dramatic decrease in ATP production by oxidative phosphorylation after silencing expression of each of these proteins while substrate phosphorylation was not severely affected. Our procyclic T. brucei cells were sensitive to the ATP synthase inhibitor oligomycin even in the presence of glucose contrary to earlier reports. Hence, the two novel proteins appear essential for the structural organization of the functional complex and regulation of mitochondrial energy generation in these organisms is more complicated than previously thought

Novel Naphthalene-Based Inhibitors of Trypanosoma brucei RNA Editing Ligase 1

African sleeping sickness is a devastating disease that plagues sub-Saharan Africa. Neglected tropical diseases like African sleeping sickness cause significant death and suffering in the world's poorest countries. Current treatments for African sleeping sickness either have high costs, terrible side effects, or limited effectiveness. Consequently, new medicines are urgently needed. RNA editing ligase 1 is an important protein critical for the survival of Trypanosoma brucei, the unicellular parasite that causes African sleeping sickness. In this paper, we describe our recent efforts to use advanced computer techniques to identify chemicals predicted to prevent RNA editing ligase 1 from functioning properly. We subsequently tested our predicted chemicals and confirmed that a number of them inhibited the protein's function. Additionally, one of the chemicals was effective at stopping the growth of the parasite in culture. Although substantial work remains to be done in order to optimize these chemicals so they are effective and safe to use in human patients, the identification of these parasite-killing compounds is nevertheless a valuable step towards finding a better cure for this devastating disease

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. b. rhodesiense require cyclical development in tsetse flies (genus Glossina) for transmission. In this study, we investigated the evolutionary origins of T. evansi. We used 15 polymorphic microsatellites to quantify levels and patterns of genetic diversity among 41 T. evansi isolates and 66 isolates of T. b. brucei (n = 51) and T. b. rhodesiense (n = 15), including many from Kenya, a region where T. evansi may have evolved from T. brucei. We found that T. evansi strains belong to at least two distinct T. brucei genetic units and contain genetic diversity that is similar to that in T. brucei strains. Results indicated that the 41 T. evansi isolates originated from multiple T. brucei strains from different genetic backgrounds, implying independent origins of T. evansi from T. brucei strains. This surprising finding further suggested that the acquisition of the ability of T. evansi to be transmitted mechanically, and thus the ability to escape the obligate link with the African tsetse fly vector, has occurred repeatedly. These findings, if confirmed, have epidemiological implications, as T. brucei strains from different genetic backgrounds can become either causative agents of a dangerous, cosmopolitan livestock disease or of a lethal human disease, like for T. b. rhodesiense

Diverse Effects on Mitochondrial and Nuclear Functions Elicited by Drugs and Genetic Knockdowns in Bloodstream Stage Trypanosoma brucei

The parasite Trypanosoma brucei causes human African trypanosomiasis, which is fatal unless treated. Currently used drugs are toxic, difficult to administer, and often are no longer effective due to drug resistance. The search for new drugs is long and expensive, and determining which compounds are worth pursuing is a key challenge in that process. In this study we sought to determine whether different compounds elicited different responses in the mammalian-infective stage of the parasite. We also examined whether genetic knockdown of parasite molecules led to similar responses. Our results show that, depending on the treatment, the replication of the parasite genomes, proper division of the cell, and mitochondrial function can be affected. Surprisingly, these different responses were not able to predict which compounds affected the long term proliferative potential of T. brucei. We found that some of the compounds had irreversible effects on the parasites within one day, so that even cells that appeared healthy could not proliferate. We suggest that determining which compounds set the parasites on a one-way journey to death may provide a means of identifying those that could lead to drugs with high efficacy