Search

Trypanosoma brucei is one of the most ancient eukaryotes where RNA interference (RNAi) is operational and is the only single-cell pathogen where RNAi has been extensively studied and used as a tool for functional analyses. Here, we report that the T. brucei RNAi pathway, although relying on a single Argonaute protein (AGO1), is initiated by the activities of two distinct Dicer-like enzymes. Both TbDCL1, a mostly cytoplasmic protein, and the previously undescribed nuclear enzyme TbDCL2 contribute to the biogenesis of siRNAs from retroposons. However, TbDCL2 has a predominant role in generating siRNAs from chromosomal internal repeat transcripts that accumulate at the nucleolus in RNAi-deficient cells and in initiating the endogenous RNAi response against retroposons and repeats alike. Moreover, siRNAs generated by both TbDCL1 and TbDCL2 carry a 5'-monophosphate and a blocked 3' terminus, suggesting that 3' end modification is an ancient trait of siRNAs. We thus propose a model whereby TbDCL2 fuels the T. brucei nuclear RNAi pathway and TbDCL1 patrols the cytoplasm, posttranscriptionally silencing potentially harmful nucleic acid parasites that may access the cytoplasm. Nevertheless, we also provide evidence for cross-talk between the two Dicer-like enzymes, because TbDCL2 is implicated in the generation of 35- to 65-nucleotide intermediate transcripts that appear to be substrates for TbDCL1. Our finding that dcl2KO cells are more sensitive to RNAi triggers than wild-type cells has significant implications for reverse genetic analyses in this important human pathogen. repeat-derived siRNAs | retroposons | RNase III | siRNA modification doi/ 10.1073/pnas.0907766106

In trypanosomes, the production of mRNA relies on the synthesis of the spliced leader (SL) RNA. Expression of the SL RNA is initiated at the only known RNA polymerase II promoter in these parasites. In the pathogenic trypanosome, Trypanosoma brucei, transcription factor liB (tTFIIB) is essential for SL RNA gene transcription and cell viability, but has a highly divergent primary sequence in comparison to TFIIB in well-studied eukaryotes. Here we describe the 2.3 A resolution structure of the C-terminal domain of tTFIIB ([tTFIIB.sub.c]). The [tTFIIB.sub.c] structure consists of 2 closely packed helical modules followed by a C-terminal extension of 32 aa. Using the structure as a guide, alanine substitutions of basic residues in regions analogous to functionally important regions of the well-studied eukaryotic TFIIB support conservation of a general mechanism of TFIIB function in eukaryotes. Strikingly, [tTFIIB.sub.c] contains additional loops and helices, and, in contrast to the highly basic DNA binding surface of human TFIIB, contains a neutral surface in the corresponding region. These attributes probably mediate trypanosome-specific interactions and have implications for the apparent bidirectional transcription by RNA polymerase II in protein-encoding gene expression in these organisms. eukaryotic transcription | parasite | RNA polymerase II | trypanosome

Antigenic variation allows Trypanosoma brucei to evade the host immune response by switching the expression of 1 out of ~15 telomeric variant surface glycoprotein (VSG) expression sites (ESs). VSG ES transcription is mediated by RNA polymerase I in a discrete nuclear site named the ES body (ESB). However, nothing is known about how the monoallelic VSG ES transcriptional state is maintained over generations. In this study, we show that during S and G2 phases and early mitosis, the active VSG ES locus remains associated with the single ESB and exhibits a delay in the separation of sister chromatids relative to control loci. This delay is dependent on the cohesin complex, as partial knockdown of cohesin subunits resulted in premature separation of sister chromatids of the active VSG ES. Cohesin depletion also prompted transcriptional switching from the active to previously inactive VSG ESs. Thus, in addition to maintaining sister chromatid cohesion during mitosis, the cohesin complex plays an essential role in the correct epigenetic inheritance of the active transcriptional VSG ES state.

Human African trypanosomiasis, or sleeping sickness caused by Trypanosoma brucei gambiense, occurs in Western and Central Africa. T. brucei s.I. displays a huge diversity of adaptations and host specificities, and questions about its reproductive mode, dispersal abilities, and effective size remain under debate. We have investigated genetic variation at 8 microsatellite loci of T. b. gambiense strains isolated from human African trypanosomiasis patients in the Ivory Coast and Guinea, with the aim of knowing how genetic information was partitioned within and between individuals in both temporal and spatial scales. The results indicate that (i) migration of T. b. gambiense group 1 strains does not occur at the scale of West Africa, and that even at a finer scale (e.g., within Guinea) migration is restricted; (ii) effective population sizes of trypanosomes, as reflected by infected hosts, are probably higher than what the epidemiological surveys suggest; and (iii) T. b. gambiense group 1 is most likely a strictly clonally reproducing organism. clonality | effective population size | genetic differentiation | genetic diversity | microsatellite markers

Trypanosomatid RNA editing is a unique process and essential for these organisms. It therefore represents a drug target for a group of protozoa that includes the causative agents for African sleeping sickness and other devastating tropical and subtropical diseases. Here, we present drug-like inhibitors of a key enzyme in the editing machinery, RNA-editing ligase 1 (REL1). These inhibitors were identified through a strategy employing molecular dynamics to account for protein flexibility. A virtual screen of the REL1 crystal structure against the National Cancer Institute Diversity Set was performed by using AutoDock4. The top 30 compounds, predicted to interact with REL1's ATP-binding pocket, were further refined by using the relaxed complex scheme (RCS), which redocks the compounds to receptor structures extracted from an explicitly solvated molecular dynamics trajectory. The resulting reordering of the ligands and filtering based on drug-like properties resulted in an initial recommended set of 8 ligands, 2 of which exhibited micromolar activity against REL1. A subsequent hierarchical similarity search with the most active compound over the full National Cancer Institute database and RCS rescoring resulted in an additional set of 6 ligands, 2 of which were confirmed as REL1 inhibitors with [IC.sub.50] values of [approximately equal to] 1 [micro]M. Tests of the 3 most promising compounds against the most closely related bacteriophage T4 RNA ligase 2, as well as against human DNA ligase III[beta], indicated a considerable degree of selectivity for RNA ligases. These compounds are promising scaffolds for future drug design and discovery efforts against these important pathogens. molecular dynamics | relaxed complex scheme | RNA ligase | African sleeping sickness | receptor flexibility

Trypanosoma brucei is a kinetoplastid flagellate, the agent of human sleeping sickness and ruminant nagana in Africa. Kinetoplastid flagellates contain their eponym kinetoplast DNA (kDNA), consisting of two types of interlocked circular DNA molecules: scores of maxicircles and thousands of minicircles. Maxicircles have typical mitochondrial genes, most of which are translatable only after RNA editing. Minicircles encode guide RNAs, required for decrypting the maxicircle transcripts. The life cycle of T. brucei involves a bloodstream stage (BS) in vertebrates and a procyclic stage (PS) in the tsetse fly vector. Partial [dyskinetoplastidy (Dk)] or total [akinetoplastidy (Ak)] loss of kDNA locks the trypanosome in the BS form. Transmission between vertebrates becomes mechanical without PS and tsetse mediation, allowing the parasite to spread outside the African tsetse belt. Trypanosoma equiperdum and Trypanosoma evansiare agents of dourine and surra, diseases of horses, camels, and water buffaloes. We have characterized representative strains of T. equiperdum and T. evansi by numerous molecular and classical parasitological approaches. We show that both species are actually strains of T. brucei, which lost part (Dk) or all (Ak) of their kDNA. These trypanosomes are not monophyletic clades and do not qualify for species status. They should be considered two subspecies, respectively T. brucei equiperdum and T. brucei evansi, which spontaneously arose recently. Dk/Ak trypanosomes may potentially emerge repeatedly from T. brucei. dourine | mitochondria | protozoa | RNA editing | surra

Uridine (U)-insertion/deletion RNA editing in trypanosome mitochondria involves an initial cleavage of the preedited mRNA at specific sites determined by the annealing of partially complementary guide RNAs. An involvement of two RNase Ill-containing core editing complex (L-complex) proteins, MP90 (KREPB1) and MP61 (KREPB3) in, respectively, U-deletion and U-insertion editing, has been suggested, but these putative enzymes have not been characterized or expressed in active form. Recombinant MP90 proteins from Trypanosorna brucei and Leishmania rnajorwere expressed in insect cells and cytosol of Leishmania tarentolae, respectively. These proteins were active in specifically cleaving a model U-deletion site and not a U-insertion site. Deletion or mutation of the RNase III motif abolished this activity. Full-round guide RNA (gRNA)-mediated in vitro U-deletion editing was reconstituted by a mixture of recombinant MP90 and recombinant RNA editing exonuclease I from L. major, and recombinant RNA editing RNA ligase 1 from L. tarentolae. MP90 is designated REN1, for RNA-editing nuclease 1. Leishmania | nuclease | RNase III | L-complex

RNA editing adds and deletes uridine nucleotides in many pre-edited mRNAs to create translatable mRNAs in the mitochondria of the parasite Trypanosoma brucei. Kinetoplastid RNA editing protein B3 (KREPB3, formerly TbMP61) is part of the multiprotein complex that catalyzes editing in T. brucei and contains an RNase III motif that suggests nuclease function. Repression of KREPB3 expression, either by RNA interference in procyclic forms (PFs) or by conditional inactivation of an ectopic KREPB3 allele in bloodstream forms (BFs) that lack both endogenous alleles, strongly inhibited growth and in vivo editing in PFs and completely blocked them in BFs. KREPB3 repression inhibited cleavage of insertion editing substrates but not deletion editing substrates in vitro, whereas the terminal uridylyl transferase, U-specific exoribonuclease, and ligase activities of editing were unaffected, and [approximately equal to] 20S editosomes were retained. Expression of KREPB3 alleles with single amino acid mutations in the RNase III motif had similar consequences. These data indicate that KREPB3 is an RNA editing endonuclease that is specific for insertion sites and is accordingly renamed KREN2 (kinetoplastid RNA editing endonudease 2). KREN2 | trypanosome | editosome | kinetoplastid

A comparison of gene content and genome architecture of Trypanosoma brucei, Trypanosoma cruzi, and Leishmania major, three related pathogens with different life cycles and disease pathology, revealed a conserved core proteome of about 6200 genes in large syntenic polycistronic gene clusters. Many species-specific genes, especially large surface antigen families, occur at nonsyntenic chromosome-internal and subtelomeric regions. Retroelements, structural RNAs, and gene family expansion are often associated with syntenic discontinuities that--along with gene divergence, acquisition and loss, and rearrangement within the syntenic regions--have shaped the genomes of each parasite. Contrary to recent reports, our analyses reveal no evidence that these species are descended from an ancestor that contained a photosynthetic endosymbiont., The protozoan pamogens Leishmania major, Trypanosoma cruzi, and Trypanosoma brucei (family Trypanosomatidae, order Kinetoplastida) collectively cause disease and death in millions of humans and countless infections in other mammals, primarily [...]

The [approximately equal to] 20S RNA ligase-containing complex (L-complex) in trypanosomatid mitochondria interacts by means of RNA linkers with at least two other multiprotein complexes to mediate the editing of mitochondrial cryptogene transcripts. The L-complex contains [approximately equal to] 16 proteins, including the two RNA-editing ligases (RELs), REL1 and REL2. Leishmania tarentolae REL1 and REL2 and Trypanosoma brucei REL1 were expressed as enzymatically active tandem affinity purification-tagged proteins in a Baculovirus system. When these proteins were added to mitochondrial lysates from T. brucei procyclic cells that were depleted of the cognate endogenous ligase by RNA interference down-regulation of expression, the added proteins were integrated into the L-complex, and, in the case of REL1, there was a complementation of in vitro-precleaved U-insertion and U-deletion editing activities of the 20S L-complex. Integration of the recombinant proteins did not occur or occurred at a very low level with noncognate ligase-depleted L-complex or with wild-type L-complex. A C-terminal region of the T. brucei recombinant REL1 downstream of the catalytic domain was identified as being involved in integration into the L-complex. The ability to perform functional complementation in vitro provides a powerful tool for molecular dissection of the editing reaction. editing | RNA-editing ligase 1 | RNA-editing ligase 2 | trypanosome

The tsetse fly-transmitted protozoan parasite Trypanosoma brucei is the causative agent of human African sleeping sickness and the cattle disease Nagana. The bloodstream form of the parasite uses a dense cell-surface coat of variant surface glycoprotein to escape the innate and adaptive immune responses of the mammalian host and a highly glycosylated transferrin receptor to take up host transferrin, an essential growth factor. These glycoproteins, as well as other flagellar pocket, endosomal, and lysosomal glycoproteins, are known to contain galactose. The parasite is unable to take up galactose, suggesting that it may depend on the action of UDP-glucose 4'-epimerase for the conversion of UDP-Glc to UDP-Gal and subsequent incorporation of galactose into glycoconjugates via UDP-Gal-dependent galactosyltransferases. In this paper, we describe the cloning of T. brucei galE, encoding T. brucei UDP-Glc-4'-epimerase, and functional characterization by complementation of a galE-deficient Escherichia coli mutant and enzymatic assay of recombinant protein. A tetracycline-inducible conditional galE null mutant of T. brucei was created using a transgenic parasite expressing the TETR tetracycline repressor protein gene. Withdrawal of tetracycline led to a cessation of cell division and substantial cell death, demonstrating that galactose metabolism in T. brucei proceeds via UDP-Glc-4'-epimerase and is essential for parasite growth. After several days without tetracycline, cultures spontaneously recovered. These cells were shown to have undergone a genetic rearrangement that deleted the TETR gene. The results show that enzymes and transporters involved in galactose metabolism may be considered as potential therapeutic targets against African trypanosomiasis. UDP-Gal | galE | epimerase

The mitochondrion of Trypanosoma brucei lacks tRNA genes. Its translation system therefore depends on the import of cytosolic, nucleus-encoded tRNAs. Thus, most trypanosomal tRNAs function in both the cytosol and the mitochondrion, and all are of the eukaryotic type. This is also the case for the elongator tRN[A.sup.Met], whereas the only other trypanosomal tRN[A.sup.Met], the eukaryotic initiator, is found exclusively in the cytosol. Unlike their cytosolic counterparts, organellar initiator tRN[As.sup.Met] carry a formylated methionine. This raises the question of how initiation of translation works in trypanosomal mitochondria, where only elongator tRN[A.sup.Met] is found. Using in organello charging and formylation assays, we show that unexpectedly a fraction of elongator tRN[A.sup.Met] becomes formylated after import into mitochondria. Furthermore, in vitro experiments with mitochondrial extracts demonstrate that only the trypanosomal elongator and not the initiator tRN[A.sup.Met] is recognized by the formylation activity. Finally, RNA interference assays identify the gene encoding the trypanosomal formylase activity. Whereas the predicted protein is homologous to prokaryotic and mitochondrial methionyl-tRN[A.sup.Met] formyltransferases, it has about twice the mass of any of these proteins.

A study was performed to demonstrate that the serum-resistance-associated (SRA) gene is an expression site-associated gene of the Edinburgh trypanozoon antigen type 1.10 (ETat 1.10) expression site. The SRA gene was encoded a variant surface glycoprotein-like protein and was only present in the R clones of the ETat strain of the Trypanosoma brucei rhodesiense resistant to normal human serum. Results revealed that SRA resulted to the resistance of the organism against normal human serum.

The presence of mating type genetic exchange barriers in Trypanosoma brucei was analyzed during six inter- and three intraclone mating experiments. Three out of the six Trypanosoma mating experiments produced a double drug-resistant progeny which was produced by trypanosomes derived from the salivary glands of flies. Furthermore, intraclone mating occurred only during sexual reproduction among heterologous trypanosomes which was triggered by the recognition of non-self between the clones.

We have studied the mechanism of accurate in vitro RNA editing of Trypanosoma brucei ATPase 6 mRNA, using four mRNA-guide RNA (gRNA) pairs that specify deletion of 2, 3, or 4 U residues at editing site 1 and mitochondrial extract. This extract not only catalyzes deletion of the specified number of U residues but also exhibits a novel endonuclease activity that cleaves the input pre-mRNA in a gRNA-directed manner, precisely at the phosphodiester bond predicted in a simple enzymatic model of RNA editing. This cleavage site is inconsistent with a chimera-based editing mechanism. The U residues to be deleted, present at the 3[prime] end of the upstream cleavage product, are then removed evidently by a 3[prime] U-specific exonuclease and not by a reverse reaction of terminal U transferase. RNA ligase can then join the mRNA halves through their newly formed 5[prime] P and 3[prime] OH termini, generating mRNA faithfully edited at the first editing site. This resultant, partially edited mRNA can then undergo accurate, gRNA-directed cleavage at editing site 2, again precisely as predicted by the enzymatic editing model. All of these enzymatic activities cofractionate with the U-deletion activity and may reside in a single complex. The data imply that each round of editing is a four-step process, involving (i) gRNA-directed cleavage of the pre-mRNA at the bond immediately 5[prime] of the region base paired to the gRNA, (ii) U deletion from or U addition to the 3[prime] OH of the upstream mRNA half, (iii) ligation of the mRNA halves, and (iv) formation of additional base pairing between the correctly edited site and the gRNA that directs subsequent nuclease cleavage at the next editing site.

Baculovirus expression system expresses ESAG 6 and ESAG 7 of Trypanosoma brucei transferrin-binding proteins, separately and together, in insect cells. Transferrin-binding needs this coexpression of ESAG 6 and 7 in the heterologous system. Transferrin-binding activities are observed on the cell surface.

The final developmental stage of the African trypanosome is characterized by a restricted expression in the salivary gland of variant surface glycoproteins (VSGs) to a subset containing 10 to 20 metacyclic variant antigen types (MVATs). A bloodstream trypanosome clone expressing an MVAT VSG gene was isolated and characterized. The results showed that bloodstream reexpression of this VSG gene is associated with a gene conversion event characterized by multiple point mutations. These mutations can further contribute to trypanosome diversity.

The paradigm of antigenic variation in parasites is the variant surface glycoprotein (VSG) of African trypanosomes. Only one VSG is expressed at any time, except for short periods during switching. The reasons for this pattern of expression and the consequences of expressing more than one VSG are unknown. Trypanosoma brucei was genetically manipulated to generate cell lines that expressed two VSGs simultaneously. These VSGs were produced in equal amounts and were homogeneously distributed on the trypanosome surface. The double-expressor cells had similar population doubling times and were as infective as wild-type cells. Thus, the simultaneous expression of two VSGs is not intrinsically harmful., In its bloodstream stage, Trypanosoma brucei evades the host immune response by a process of antigenic variation, in which the surface coat, consisting of about [10.sup.7] copies of a glycosylphosphatidylinositol [...]

The first functional and structural characterization of the inosine-guanosine-specific nucleoside hydrolase (NH) from Trypanosoma brucei brucei is reported. The study identified nanomolar iminoribitol-based inhibitors which could be important lead compounds for the development of novel therapeutic strategies against trypanosomal diseases.

The first enzymatic characterization of single-thioredoxin (Trx) quiescin sulfhydryl oxidase (QSOX) flavoenzymes from vertebrates which catalyzes the direct, facile, insertion of disulfide bonds into reduced unfolded proteins with the reduction of oxygen to hydrogen peroxide is reported. The single-Trx domain QSOX is observed to show remarkable similarity in enzymatic behavior to its double-Trx metazoan couterparts.

The kinetics of interactions of different regions of loops 3 surrounding the residue occupying position 14 were studied to determine the stability of homodimeric glycolytic enzyme triosephosphate isomerase from Trypanosoma brucei (TbTIM). The findings suggest that the residues that constitute the portion of the interface formed by loop 3 and residue 14 of the other monomer, the amino acid at position 79, are critically fundamental to maintain TbTIM enzyme stability.

The structures of Trypanosoma brucei apoMRP1/MRP2 complex and an MRP1/MRP2-gRNA complex are determined to understand the mechanism by which this complex performs RNA matchmaking. The gRNA molecule binds to the highly basic [beta] sheet surface of the MRP complex via non-specific, electrostatic contacts while the gRNA stem/loop II base is anchored to the basic surface, stem/loop I which is unfolded and exposed to solvent.

The African trypanosome, Trypanosoma brucei, has been shown to undergo genetic exchange in the laboratory, but controversy exists as to the role of genetic exchange in natural populations. Much of the analysis to date has been derived from isoenzyme or randomly amplified polymorphic DNA data with parasite material from a range of hosts and geographical locations. These markers fail to distinguish between the human infective (T. b. rhodesiense) and nonhuman infective (T. b. brucei) 'subspecies' so that parasites derived from hosts other than humans potentially contain both subspecies. To overcome some of the inherent problems with the use of such markers and diverse populations, we have analyzed a well-defined population from a discrete geographical location (Busoga, Uganda) using three recently described minisatellite markers. The parasites were primarily isolated from humans and cattle with the latter isolates further characterized by their ability to resist lysis by human serum (equivalent to human infectivity). The minisatellite markers show high levels of polymorphism, and from the data obtained we conclude that T. b. rhodesiense is genetically isolated from T. b. brucei and can be unambiguously identified by its multilocus genotype. Analysis of the genotype frequencies in the separated T. b. brucei and T. b. rhodesiense populations shows the former has an epidemic population structure whereas the latter is clonal This finding suggests that the strong linkage disequilibrium observed in previous analyses, where human and nonhuman infective trypanosomes were not distinguished, results from the treatment of two genetically isolated populations as a single population.