Parasitic Protozoan Trypanosoma Brucei Research Articles

Dihydrofolate reductase and membrane translocation: evolution of a classic experiment: Classic landmark papers, irrespective of their age, can teach students how best science is practiced and inspire new experiments.

Any PhD student or postdoc, who scrambles to write and submit a publication before getting scooped, can testify that science is a fast‐moving endeavor. Given our limited time and the ever‐increasing pace with which scientific studies are published, few students and postdocs—and PIs as a matter of fact—have time to keep up with the literature. “Reading” a manuscript often means just skimming through the abstract, having a quick glimpse at the figures and searching the PDF file for keywords of immediate interest. Notwithstanding these constraints, I would argue that the scientific literature is a treasure trove of information and ideas that go beyond contemporary papers to include classical publications. However, it can be difficult to motivate students and postdocs to read old landmark publications that reported groundbreaking discoveries and opened up new avenues of research. Many think that these papers are of merely historic interest and have little to contribute to scientists expected to use cutting‐edge methods to produce high‐impact publications. > Reading a manuscript often means just skimming through the abstract, having a quick glimpse at the figures and searching the PDF file for keywords of immediate interest. However, classic papers still have a lot to offer. For once, they withstood the test of the time, as the reported results have shown to be correct and reproducible. This is unfortunately not the case for many high‐impact publications today, which, owing to the hype of selling it to the highest impact factor journal, just tell a cool story that in reality may be much less clear‐cut than reported. Furthermore, classic scientific publications often impress by a conceptual clarity and in many cases simplicity and thereby offer valuable lessons of how best science should be practiced. These are qualities I miss in many of today's papers that contain exceedingly large data sets …

Biogenesis of the mitochondrial DNA inheritance machinery in the mitochondrial outer membrane of Trypanosoma brucei.

Mitochondria cannot form de novo but require mechanisms that mediate their inheritance to daughter cells. The parasitic protozoan Trypanosoma brucei has a single mitochondrion with a single-unit genome that is physically connected across the two mitochondrial membranes with the basal body of the flagellum. This connection, termed the tripartite attachment complex (TAC), is essential for the segregation of the replicated mitochondrial genomes prior to cytokinesis. Here we identify a protein complex consisting of three integral mitochondrial outer membrane proteins—TAC60, TAC42 and TAC40—which are essential subunits of the TAC. TAC60 contains separable mitochondrial import and TAC-sorting signals and its biogenesis depends on the main outer membrane protein translocase. TAC40 is a member of the mitochondrial porin family, whereas TAC42 represents a novel class of mitochondrial outer membrane β-barrel proteins. Consequently TAC40 and TAC42 contain C-terminal β-signals. Thus in trypanosomes the highly conserved β-barrel protein assembly machinery plays a major role in the biogenesis of its unique mitochondrial genome segregation system.

Design and Synthesis of Brain Penetrant Trypanocidal N-Myristoyltransferase Inhibitors.

N-Myristoyltransferase (NMT) represents a promising drug target within the parasitic protozoa Trypanosoma brucei (T. brucei), the causative agent for human African trypanosomiasis (HAT) or sleeping sickness. We have previously validated T. brucei NMT as a promising druggable target for the treatment of HAT in both stages 1 and 2 of the disease. We report on the use of the previously reported DDD85646 (1) as a starting point for the design of a class of potent, brain penetrant inhibitors of T. brucei NMT.

TRNAs and proteins use the same import channel for translocation across the mitochondrial outer membrane of trypanosomes

Mitochondrial tRNA import is widespread, but the mechanism by which tRNAs are imported remains largely unknown. The mitochondrion of the parasitic protozoan Trypanosoma brucei lacks tRNA genes, and thus imports all tRNAs from the cytosol. Here we show that in T. brucei in vivo import of tRNAs requires four subunits of the mitochondrial outer membrane protein translocase but not the two receptor subunits, one of which is essential for protein import. The latter shows that it is possible to uncouple mitochondrial tRNA import from protein import. Ablation of the intermembrane space domain of the translocase subunit, archaic translocase of the outer membrane (ATOM)14, on the other hand, while not affecting the architecture of the translocase, impedes both protein and tRNA import. A protein import intermediate arrested in the translocation channel prevents both protein and tRNA import. In the presence of tRNA, blocking events of single-channel currents through the pore formed by recombinant ATOM40 were detected in electrophysiological recordings. These results indicate that both types of macromolecules use the same import channel across the outer membrane. However, while tRNA import depends on the core subunits of the protein import translocase, it does not require the protein import receptors, indicating that the two processes are not mechanistically linked.

Mitochondrial protein import in trypanosomes: Expect the unexpected.

Mitochondria have many different functions, the most important one of which is oxidative phosphorylation. They originated from an endosymbiotic event between a bacterium and an archaeal host cell. It was the evolution of a protein import system that marked the boundary between the endosymbiotic ancestor of the mitochondrion and a true organelle that is under the control of the nucleus. In present day mitochondria more than 95% of all proteins are imported from the cytosol in a proces mediated by hetero-oligomeric protein complexes in the outer and inner mitochondrial membranes. In this review we compare mitochondrial protein import in the best studied model system yeast and the parasitic protozoan Trypanosoma brucei. The 2 organisms are phylogenetically only remotely related. Despite the fact that mitochondrial protein import has the same function in both species, only very few subunits of their import machineries are conserved. Moreover, while yeast has 2 inner membrane protein translocases, one specialized for presequence-containing and one for mitochondrial carrier proteins, T. brucei has a single inner membrane translocase only, that mediates import of both types of substrates. The evolutionary implications of these findings are discussed.

Mitochondrial protein import - Functional analysis of the highly diverged Tom22 orthologue of Trypanosoma brucei

The β-barrel protein Tom40 and the α-helically anchored membrane protein Tom22 are the only universally conserved subunits of the protein translocase of the mitochondrial outer membrane (TOM). Tom22 has an N-terminal cytosolic and a C-terminal intermembrane space domain. It occurs in two variants: one typified by the yeast protein which has a cytosolic domain containing a cluster of acidic residues, and a shorter variant typified by the plant protein that lacks this domain. Yeast-type Tom22 functions as a secondary protein import receptor and is also required for the stability of the TOM complex. Much less is known about the more widespread short variant of Tom22, which is also found in the parasitic protozoan Trypanosoma brucei. Here we show that the intermembrane space domain of trypanosomal Tom22 binds mitochondrial precursor proteins and that it is essential for normal growth and mitochondrial protein import. Moreover, complementation experiments indicate that the intermembrane space domain cannot be replaced by the corresponding regions of the yeast or plant Tom22 orthologues. Lack or replacement of the short cytosolic domain, however, does not interfere with protein function. Finally, we show that only the membrane-spanning domain of trypanosomal Tom22 is essential for assembly of the trypanosomal TOM complex analogue.

RFT1 Protein Affects Glycosylphosphatidylinositol (GPI) Anchor Glycosylation

The membrane protein RFT1 is essential for normal protein N-glycosylation, but its precise function is not known. RFT1 was originally proposed to translocate the glycolipid Man5GlcNAc2-PP-dolichol (needed to synthesize N-glycan precursors) across the endoplasmic reticulum membrane, but subsequent studies showed that it does not play a direct role in transport. In contrast to the situation in yeast, RFT1 is not essential for growth of the parasitic protozoan Trypanosoma brucei, enabling the study of its function in a null background. We now report that lack of T. brucei RFT1 (TbRFT1) not only affects protein N-glycosylation but also glycosylphosphatidylinositol (GPI) anchor side-chain modification. Analysis by immunoblotting, metabolic labeling, and mass spectrometry demonstrated that the major GPI-anchored proteins of T. brucei procyclic forms have truncated GPI anchor side chains in TbRFT1 null parasites when compared with wild-type cells, a defect that is corrected by expressing a tagged copy of TbRFT1 in the null background. In vivo and in vitro labeling experiments using radiolabeled GPI precursors showed that GPI underglycosylation was not the result of decreased formation of the GPI precursor lipid or defective galactosylation of GPI intermediates in the endoplasmic reticulum, but rather due to modifications that are expected to occur in the Golgi apparatus. Unexpectedly, immunofluorescence microscopy localized TbRFT1 to both the endoplasmic reticulum and the Golgi, consistent with the proposal that TbRFT1 plays a direct or indirect role in GPI anchor glycosylation in the Golgi apparatus. Our results implicate RFT1 in a wider range of glycosylation processes than previously appreciated.

Maf1 is a negative regulator of transcription in Trypanosoma brucei.

RNA polymerase III (Pol III) produces small RNA molecules that play essential roles in mRNA processing and translation. Maf1, originally described as a negative regulator of Pol III transcription, has been studied from yeast to human. Here we characterized Maf1 in the parasitic protozoa Trypanosoma brucei (TbMaf1), representing the first report to analyse Maf1 in an early-diverged eukaryote. While Maf1 is generally encoded by a single-copy gene, the T. brucei genome contains two almost identical TbMaf1 genes. The TbMaf1 protein has the three conserved sequences and is predicted to fold into a globular structure. Unlike in yeast, TbMaf1 localizes to the nucleus in procyclic forms of T. brucei under normal growth conditions. Cell lines that either downregulate or overexpress TbMaf1 were generated, and growth curve analysis with them suggested that TbMaf1 participates in the regulation of cell growth of T. brucei. Nuclear run-on and chromatin immunoprecipitation analyses demonstrated that TbMaf1 represses Pol III transcription of tRNA and U2 snRNA genes by associating with their promoters. Interestingly, 5S rRNA levels do not change after TbMaf1 ablation or overexpression. Notably, our data also revealed that TbMaf1 regulates Pol I transcription of procyclin gene and Pol II transcription of SL RNA genes.

The parasitic protozoan Trypanosoma brucei contains a non canonical protein translocase in the outer membrane of its mitochondrion. A 3D reconstruction of the insect stage form of the parasite (obtained by serial block face scanning electron microscopy) i

Molecular MicrobiologyVolume 102, Issue 3 p. i-i Front CoverFree Access The parasitic protozoan Trypanosoma brucei contains a non canonical protein translocase in the outer membrane of its mitochondrion. A 3D reconstruction of the insect stage form of the parasite (obtained by serial block face scanning electron microscopy) illustrates the network-like mitochondrion (green), basal body (white), flagellum (purple) and DNA (blue). Image courtesy: Jiří Týč and Sue Vaughan (Oxford Brookes University, Oxford, UK). For details see article by Desy et al. on pp. 520–529 of this issue. First published: 21 October 2016 https://doi.org/10.1111/mmi.13195AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume102, Issue3November 2016Pages i-i RelatedInformation

The nuclear envelope and gene organization in parasitic protozoa: Specializations associated with disease

The parasitic protozoa Trypanosoma brucei and Plasmodium falciparum are lethal human parasites that have developed elegant strategies of immune evasion by antigenic variation. Despite the vast evolutionary distance between the two taxa, both parasites employ strict monoallelic expression of their membrane proteins, variant surface glycoproteins in Trypanosomes and the var, rif and stevor genes in Plasmodium, in order to evade their host’s immune system. Additionally, both telomeric location and epigenetic controls are prominent features of these membrane proteins. As such, telomeres, chromatin structure and nuclear organization all contribute to control of gene expression and immune evasion. Here, we discuss the importance of epigenetics and sub-nuclear context for the survival of these disease-causing parasites.

Pseudoenzymes as Activators of Enzyme Function: The Prozyme Paradigm in Trypanosomatid Polyamine Metabolism

The parasitic protozoa Trypanosoma brucei is the causative agent of human African trypanosomiasis (HAT). Polyamine metabolism is a proven drug target for the treatment of HAT as eflornithine, an inhibitor of the key biosynthetic enzyme ornithine decarboxylase, is a frontline therapy for late stage disease. Our studies have uncovered an exciting and novel mechanism for regulation of polyamine biosynthesis in the trypanosomatids. We have demonstrated that two enzymes in the pathway, S‐adenosylmethinione decarboxylase (AdoMetDC) and deoxyhypusine synthase (DHS), are activated by oligomerization with catalytically dead paralogs we term prozymes. The trypanosomatids are the only organisms where this mechanism is used to generate the functional forms of both of these enzymes, and while all metazoan genomes contain evidence of “pseudo enzymes”, some with regulatory roles, the sheer magnitude of the enzymatic activation (1000‐3000‐fold) observed for these trypanosomatid enzymes is unprecedented. Additionally in the case of AdoMetDC, changes in AdoMetDC activity levels in the parasite lead to increases in the prozyme protein levels, correlated to the levels of decarboxylated AdoMet, suggesting that regulation of prozyme expression is a novel mechanism for controlling polyamine levels in these parasites. Our finding that this mechanism evolved twice within the same pathway also suggests that the trypanosomatids are under selective pressure to evolve novel mechanisms of regulation, perhaps because of the lack of transcriptional and other mechanisms typically used in eukaryotes to regulate gene expression.

Discovery of an ergosterol-signaling factor that regulates Trypanosoma brucei growth

Ergosterol biosynthesis and homeostasis in the parasitic protozoan Trypanosoma brucei was analyzed by RNAi silencing and inhibition of sterol C24β-methyltransferase (TbSMT) and sterol 14α-demethylase [TbSDM (TbCYP51)] to explore the functions of sterols in T. brucei growth. Inhibition of the amount or activity of these enzymes depletes ergosterol from cells at <6 fg/cell for procyclic form (PCF) cells or <0.01 fg/cell for bloodstream form (BSF) cells and reduces infectivity in a mouse model of infection. Silencing of TbSMT expression by RNAi in PCF or BSF in combination with 25-azalanosterol (AZA) inhibited parasite growth and this inhibition was restored completely by adding synergistic cholesterol (7.8 μM from lipid-depleted media) with small amounts of ergosterol (1.2 μM) to the medium. These observations are consistent with the proposed requirement for ergosterol as a signaling factor to spark cell proliferation while imported cholesterol or the endogenously formed cholesta-5,7,24-trienol act as bulk membrane components. To test the potential chemotherapeutic importance of disrupting ergosterol biosynthesis using pairs of mechanism-based inhibitors that block two enzymes in the post-squalene segment, parasites were treated with AZA and itraconazole at 1 μM each (ED50 values) resulting in parasite death. Taken together, our results demonstrate that the ergosterol pathway is a prime drug target for intervention in T. brucei infection.

Iron and Parasites

Iron and Parasites

Crystal structure of an arginase-like protein from Trypanosoma brucei that evolved without a binuclear manganese cluster.

The X-ray crystal structure of an arginase-like protein from the parasitic protozoan Trypanosoma brucei, designated TbARG, is reported at 1.80 and 2.38 Å resolution in its reduced and oxidized forms, respectively. The oxidized form of TbARG is a disulfide-linked hexamer that retains the overall architecture of a dimer of trimers in the reduced form. Intriguingly, TbARG does not contain metal ions in its putative active site, and amino acid sequence comparisons indicate that all but one of the residues required for coordination to the catalytically obligatory binuclear manganese cluster in other arginases are substituted here with residues incapable of metal ion coordination. Therefore, the structure of TbARG is the first of a member of the arginase/deacetylase superfamily that is not a metalloprotein. Although we show that metal binding activity is easily reconstituted in TbARG by site-directed mutagenesis and confirmed in X-ray crystal structures, it is curious that this protein and its parasitic orthologues evolved away from metal binding function. Knockout of the TbARG gene from the genome demonstrated that its function is not essential to cultured bloodstream-form T. brucei, and metabolomics analysis confirmed that the enzyme has no role in the conversion of l-arginine to l-ornithine in these cells. While the molecular function of TbARG remains enigmatic, the fact that the T. brucei genome encodes only this protein and not a functional arginase indicates that the parasite must import l-ornithine from its host to provide a source of substrate for ornithine decarboxylase in the polyamine biosynthetic pathway, an active target for the development of antiparasitic drugs.

Involvement of a type 2 ribonuclease H in kinetoplast maxicircle DNA replication in Trypanosoma brucei (LB165)

Ribonuclease H (RNase H, EC # 3.1.26.4) catalyzes the hydrolysis of RNA from a DNA/RNA hybrid. This ubiquitous enzyme interacts with several different DNA‐modifying enzymes in the cell and participates in the replication, recombination and repair of nuclear and mitochondrial DNA molecules. We report here that the type 2 RNase H of the parasitic protozoan Trypanosoma brucei is a predominantly mitochondrial enzyme and is essential for the survival of both the procyclic and the blood stream forms. Yeast 2‐hybrid and co‐IP analyses for the mitochondrial DNA replication‐associated proteins interacting directly with RNase HIIC revealed binding of RNase HIIC with the mitochondrial DNA ligase Kα which is involved in kinetoplast DNA replication. Interestingly, we found that RNase HIIC also binds with the DNA recombinase DMC1 which is also located in the mitochondrion of the parasite. Chromatin immunoprecipitation data indicated binding of both RNase HIIC and DMC1 to the maxicircle DNA. We found that the maxicircle DNA and transcripts derived from it are decreased significantly in the cells with decreased levels of RNase HIIC. This enzyme is required for the recovery from DNA damage caused by caffeine, hydroxyurea and methylmethane sulfonate, as the RNase HIIC knocked down cells are more sensitive to the lethality of these DNA damaging agents. We conclude that RNase HIIC collaborates with DNA ligase Kα and DMC1 to participate in the replication and repair of mitochondrial maxicircle DNA in these parasite cells. This research was supported in parts by NIAID grants 5R01AI042327, 1R21AI076757‐01A1 and NIGMS grant #3 S06 GM008037‐34‐S2 to GC.

The emerging role of RNA-binding proteins in the life cycle ofTrypanosoma brucei

One of the key questions in understanding the biology of an organism is how to correlate cellular fate and function with gene expression patterns. This is particularly relevant for pathogenic organisms, like the parasitic protozoa Trypanosoma brucei, who often cycle between different hosts, thereby encountering vastly different environments. Survival in and adaptation to new surroundings requires activation of specific gene networks, which is most often achieved by regulatory mechanisms embedded in the transcriptional machinery. However, in T. brucei and related trypanosomatids these responses appear to be accomplished mainly by post-transcriptional mechanisms. Although an understanding of how this parasite modulates gene regulatory networks is in the early stages, RNA-binding proteins (RBPs) are beginning to take centre stage. Here, we discuss recent progress in the identification of RBPs with crucial roles in different stages of the T. brucei life cycle, and in elucidating targets of RBPs.

Mitochondrial translation factors of Trypanosoma brucei: elongation factor‐Tu has a unique subdomain that is essential for its function

Mitochondrial translation in the parasitic protozoan Trypanosoma brucei relies on imported eukaryotic-type tRNAs as well as on bacterial-type ribosomes that have the shortest known rRNAs. Here we have identified the mitochondrial translation elongation factors EF-Tu, EF-Ts, EF-G1 and release factor RF1 of trypanosomatids and show that their ablation impairs growth and oxidative phosphorylation. In vivo labelling experiments and a SILAC-based analysis of the global proteomic changes induced by EF-Tu RNAi directly link EF-Tu to mitochondrial translation. Moreover, EF-Tu RNAi reveals downregulation of many nuclear encoded subunits of cytochrome oxidase as well as of components of the bc1-complex, whereas most cytosolic ribosomal proteins were upregulated. Interestingly, T. brucei EF-Tu has a 30-amino-acid-long, highly charged subdomain, which is unique to trypanosomatids. A combination of RNAi and complementation experiments shows that this subdomain is essential for EF-Tu function, but that it can be replaced by a similar sequence found in eukaryotic EF-1a, the cytosolic counterpart of EF-Tu. A recent cryo-electron microscopy study revealed that trypanosomatid mitochondrial ribosomes have a unique intersubunit space that likely harbours the EF-Tu binding site. These findings suggest that the trypanosomatid-specific EF-Tu subdomain serves as an adaption for binding to these unusual mitochondrial ribosomes.

Product feedback regulation implicated in translational control of the Trypanosoma brucei S‐adenosylmethionine decarboxylase regulatory subunit prozyme

Human African sleeping sickness (HAT) is caused by the parasitic protozoan Trypanosoma brucei. Polyamine biosynthesis is an important drug target in the treatment of HAT. Previously we showed that trypanosomatid S-adenosylmethionine decarboxylase (AdoMetDC), a key enzyme for biosynthesis of the polyamine spermidine, is activated by heterodimer formation with an inactive paralogue termed prozyme. Furthermore, prozyme protein levels were regulated in response to reduced AdoMetDC activity. Herein we show that T. brucei encodes three prozyme transcripts. The 3'UTRs of these transcripts were mapped and chloramphenicol acetyltransferase (CAT) reporter constructs were used to identify a 1.2 kb region that contained a 3'UTR prozyme regulatory element sufficient to upregulate CAT protein levels (but not RNA) upon AdoMetDC inhibition, supporting the hypothesis that prozyme expression is regulated translationally. To gain insight into trans-acting factors, genetic rescue of AdoMetDC RNAi knock-down lines with human AdoMetDC was performed leading to rescue of the cell growth block, and restoration of prozyme protein to wild-type levels. Metabolite analysis showed that prozyme protein levels were inversely proportional to intracellular levels of decarboxylated AdoMet (dcAdoMet). These data suggest that prozyme translation may be regulated by dcAdoMet, a metabolite not previously identified to play a regulatory role.

Identification of a novel lipin homologue from the parasitic protozoan Trypanosoma brucei

BackgroundArginine methylation is a post-translational modification that expands the functional diversity of proteins. Kinetoplastid parasites contain a relatively large group of protein arginine methyltransferases (PRMTs) compared to other single celled eukaryotes. Several T. brucei proteins have been shown to serve as TbPRMT substrates in vitro, and a great number of proteins likely to undergo methylation are predicted by the T. brucei genome. This indicates that a large number of proteins whose functions are modulated by arginine methylation await discovery in trypanosomes. Here, we employed a yeast two-hybrid screen using as bait the major T. brucei type I PRMT, TbPRMT1, to identify potential substrates of this enzyme.ResultsWe identified a protein containing N-LIP and C-LIP domains that we term TbLpn. These domains are usually present in a family of proteins known as lipins, and involved in phospholipid biosynthesis and gene regulation. Far western and co-immunoprecipitation assays confirmed the TbPRMT1-TbLpn interaction. We also demonstrated that TbLpn is localized mainly to the cytosol, and is methylated in vivo. In addition, we showed that, similar to mammalian and yeast proteins with N-LIP and C-LIP domains, recombinant TbLpn exhibits phosphatidic acid phosphatase activity, and that two conserved aspartic acid residues present in the C-LIP domain are critical for its enzymatic activity.ConclusionsThis study reports the characterization of a novel trypanosome protein and provides insight into its enzymatic activity and function in phospholipid biosynthesis. It also indicates that TbLpn functions may be modulated by arginine methylation.

Physiological interaction of BRCA2 and PARP in the protozoan pathogen Trypanosoma brucei

Many eukaryotes including the parasitic protozoan Trypanosoma brucei depend heavily on the BRCA2 protein, which is important for the mediation of DNA recombination and repair. An interesting aspect of BRCA2 deficiency in cancer cells is that these cells are ultra‐sensitive to the inhibitors of the chromatin modifying enzyme poly(ADP‐ribose) polymerase (PARP). In the absence of PARP, spontaneous single‐strand breaks collapse replication forks and trigger homologous recombination for repair. Thus, PARP activity is essential in homologous recombination‐deficient BRCA2‐mutant cancer cells. BRCA2 and PARP are not only expressed in the mammalian cells but also present in high levels in many other eukaryotic cells including the parasitic protozoan T. brucei. To evaluate the universality of the importance of these two proteins in the management of DNA damage, we determined the effect of PARP inhibitors such as 3‐AB, Veliparib, PF‐01367338, Olaparib and Iniparib in BRCA2 knocked down T. brucei cells treated with the DNA damaging agent cisplatin. Our in vitro data strongly suggest that a combination of a BRCA2 inhibitor (e.g. BRCA2 BRC peptide), a PARP inhibitor and a DNA damaging agent (e.g. cisplatin) will be a chemical regimen for the management of trypanosomiasis and similar protozoan infections. Supported by NIH grants 2SC1GM081146‐05 to MC, 1U54RR026140‐01 to SM and NIH grants R01AI042327, R21AI076757 to GC.