Cyclical Transmission of Trypanosoma brucei rhodesiense and Trypanosoma congolense by Tsetse Flies Infected with Culture-Form Procyclic Trypanosomes*

SYNOPSIS. Culture procyclic forms of Trypanosoma brucei rhodesiense and Trypanosoma congolense were fed to Glossina morsitans morsitans through artificial membranes. A very high percentage of the flies so fed produced established midgut infections, a proportion of which went on to develop into mature metacyclic trypanosomes capable of infecting mammalian hosts. The method offers a safe, clean way of infecting tsetse flies with African trypanosomes which reduces the need for trypanosome-infected animals in the laboratory.     

Crystal structure of the small GTPase Arl6/BBS3 from Trypanosoma brucei

Arl6/BBS3 is a small GTPase, mutations in which are implicated in the human ciliopathy Bardet–Biedl Syndrome (BBS). Arl6 is proposed to facilitate the recruitment of a large protein complex known as the BBSome to the base of the primary cilium, mediating specific trafficking of molecules to this important sensory organelle. Orthologues of Arl6 and the BBSome core subunits have been identified in the genomes of trypanosomes. Flagellum function and motility are crucial to the survival of Trypanosoma brucei, the causative agent of human African sleeping sickness, in the human bloodstream stage of its lifecycle and so the function of the BBSome proteins in trypanosomes warrants further study. RNAi knockdown of T. brucei Arl6 (TbArl6) has recently been shown to result in shortening of the trypanosome flagellum. Here we present the crystal structure of TbArl6 with the bound non‐hydrolysable GTP analog GppNp at 2.0 Å resolution and highlight important differences between the trypanosomal and human proteins. Analysis of the TbArl6 active site confirms that it lacks the key glutamine that activates the nucleophile during GTP hydrolysis in other small GTPases. Furthermore, the trypanosomal proteins are significantly shorter at their N‐termini suggesting a different method of membrane insertion compared to humans. Finally, analysis of sequence conservation suggests two surface patches that may be important for protein–protein interactions. Our structural analysis thus provides the basis for future biochemical characterisation of this important family of small GTPases.     

Structural characterization and epitope mapping of the glutamic acid/alanine-rich protein from Trypanosoma congolense: defining assembly on the parasite cell surface

Trypanosoma congolense is an African trypanosome that causes serious disease in cattle in Sub-Saharan Africa. The four major life cycle stages of T. congolense can be grown in vitro, which has led to the identification of several cell-surface molecules expressed on the parasite during its transit through the tsetse vector. One of these, glutamic acid/alanine-rich protein (GARP), is the first expressed on procyclic forms in the tsetse midgut and is of particular interest because it replaces the major surface coat molecule of bloodstream forms, the variant surface glycoprotein (VSG) that protects the parasite membrane, and is involved in antigenic variation. Unlike VSG, however, the function of GARP is not known, which necessarily limits our understanding of parasite survival in the tsetse. Toward establishing the function of GARP, we report its three-dimensional structure solved by iodide phasing to a resolution of 1.65 Å. An extended helical bundle structure displays an unexpected and significant degree of homology to the core structure of VSG, the only other major surface molecule of trypanosomes to be structurally characterized. Immunofluorescence microscopy and immunoaffinity-tandem mass spectrometry were used in conjunction with monoclonal antibodies to map both non-surface-disposed and surface epitopes. Collectively, these studies enabled us to derive a model describing the orientation and assembly of GARP on the surface of trypanosomes. The data presented here suggest the possible structure-function relationships involved in replacement of the bloodstream form VSG by GARP as trypanosomes differentiate in the tsetse vector after a blood meal.     

Independent Pathways Can Transduce the Life-Cycle Differentiation Signal in Trypanosoma brucei

African trypanosomes cause disease in humans and livestock, generating significant health and welfare problems throughout sub-Saharan Africa. When ingested in a tsetse fly bloodmeal, trypanosomes must detect their new environment and initiate the developmental responses that ensure transmission. The best-established environmental signal is citrate/cis aconitate (CCA), this being transmitted through a protein phosphorylation cascade involving two phosphatases: one that inhibits differentiation (TbPTP1) and one that activates differentiation (TbPIP39). Other cues have been also proposed (mild acid, trypsin exposure, glucose depletion) but their physiological relevance and relationship to TbPTP1/TbPIP39 signalling is unknown. Here we demonstrate that mild acid and CCA operate through TbPIP39 phosphorylation, whereas trypsin attack of the parasite surface uses an alternative pathway that is dispensable in tsetse flies. Surprisingly, glucose depletion is not an important signal. Mechanistic analysis through biophysical methods suggests that citrate promotes differentiation by causing TbPTP1 and TbPIP39 to interact.     

Trypanosoma brucei s.l.: Microsatellite markers revealed high level of multiple genotypes in the mid-guts of wild tsetse flies of the Fontem sleeping sickness focus of Cameroon

To identify Trypanosoma brucei genotypes which are potentially transmitted in a sleeping sickness focus, microsatellite markers were used to characterize T. brucei found in the mid-guts of wild tsetse flies of the Fontem sleeping sickness focus in Cameroon. For this study, two entomological surveys were performed during which 2685 tsetse flies were collected and 1596 (59.2%) were dissected. Microscopic examination revealed 1.19% (19/1596) mid-gut infections with trypanosomes; the PCR method identified 4.7% (75/1596) infections with T. brucei in the mid-guts. Of these 75 trypanosomes identified in the mid-guts, Trypanosoma brucei gambiense represented 0.81% (13/1596) of them, confirming the circulation of human infective parasite in the Fontem focus. Genetic characterization of the 75 T. brucei samples using five microsatellite markers revealed not only multiple T. brucei genotypes (47%), but also single genotypes (53%) in the mid-guts of the wild tsetse flies. These results show that there is a wide range of trypanosome genotypes circulating in the mid-guts of wild tsetse flies from the Fontem sleeping sickness focus. They open new avenues to undertake investigations on the maturation of multiple infections observed in the tsetse fly mid-guts. Such investigations may allow to understand how the multiple infections evolve from the tsetse flies mid-guts to the salivary glands and also to understand the consequence of these evolutions on the dynamic (which genotype is transmitted to mammals) of trypanosomes transmission.     

NMR structure of the calflagin Tb24 flagellar calcium binding protein of Trypanosoma brucei

Flagellar calcium binding proteins are expressed in a variety of trypanosomes and are potential drug targets for Chagas disease and African sleeping sickness. The flagellar calcium binding protein calflagin of Trypanosoma brucei (called Tb24) is a myristoylated and palmitoylated EF‐hand protein that is targeted to the inner leaflet of the flagellar membrane. The Tb24 protein may also interact with proteins on the membrane surface that may be different from those bound to flagellar calcium binding proteins (FCaBPs) in T. cruzi. We report here the NMR structure of Tb24 that contains four EF‐hand motifs bundled in a compact arrangement, similar to the overall fold of T. cruzi FCaBP (RMSD = 1.0 Å). A cluster of basic residues (K22, K25, K31, R36, and R38) located on a surface near the N‐terminal myristoyl group may be important for membrane binding. Non‐conserved residues on the surface of a hydrophobic groove formed by EF2 (P91, Q95, D103, and V108) and EF4 (C194, T198, K199, Q202, and V203) may serve as a target protein binding site and could have implications for membrane target recognition.     

Mitochondrial pyruvate carrier in Trypanosoma brucei

Pyruvate is a key product of glycolysis that regulates the energy metabolism of cells. In Trypanosoma brucei, the causative agent of sleeping sickness, the fate of pyruvate varies dramatically during the parasite life cycle. In bloodstream forms, pyruvate is mainly excreted, whereas in tsetse fly forms, pyruvate is metabolized in mitochondria yielding additional ATP molecules. The character of the molecular machinery that mediates pyruvate transport across mitochondrial membrane was elusive until the recent discovery of mitochondrial pyruvate carrier (MPC) in yeast and mammals. Here, we characterized pyruvate import into mitochondrion of T. brucei. We identified mpc1 and mpc2 homologs in the T. brucei genome with attributes of MPC protein family and we demonstrated that both proteins are present in the mitochondrial membrane of the parasite. Investigations of mpc1 or mpc2 gene knock‐out cells proved that T. brucei MPC1/2 proteins facilitate mitochondrial pyruvate transport. Interestingly, MPC is expressed not only in procyclic trypanosomes with fully activated mitochondria but also in bloodstream trypanosomes in which most of pyruvate is excreted. Moreover, MPC appears to be essential for bloodstream forms, supporting the recently emerging picture that the functions of mitochondria in bloodstream forms are more diverse than it was originally thought.     

The essential neutral sphingomyelinase is involved in the trafficking of the variant surface glycoprotein in the bloodstream form of Trypanosoma brucei

Sphingomyelin is the main sphingolipid in Trypanosoma brucei, the causative agent of African sleeping sickness. In vitro and in vivo characterization of the T. brucei neutral sphingomyelinase demonstrates that it is directly involved in sphingomyelin catabolism. Gene knockout studies in the bloodstream form of the parasite indicate that the neutral sphingomyelinase is essential for growth and survival, thus highlighting that the de novo biosynthesis of ceramide is unable to compensate for the loss of sphingomyelin catabolism. The phenotype of the conditional knockout has given new insights into the highly active endocytic and exocytic pathways in the bloodstream form of T. brucei. Hence, the formation of ceramide in the endoplasmic reticulum affects post‐Golgi sorting and rate of deposition of newly synthesized GPI‐anchored variant surface glycoprotein on the cell surface. This directly influences the corresponding rate of endocytosis, via the recycling endosomes, of pre‐existing cell surface variant surface glycoprotein. The trypanosomes use this coupled endocytic and exocytic mechanism to maintain the cell density of its crucial variant surface glycoprotein protective coat. TbnSMase is therefore genetically validated as a drug target against African trypanosomes, and suggests that interfering with the endocytic transport of variant surface glycoprotein is a highly desirable strategy for drug development against African trypanosomasis.     

Trypanosomatid Pin1‐Type Peptidyl‐Prolyl Isomerase Is Cytosolic and Not Essential for Cell Proliferation

Pin1‐type peptidyl‐prolyl cis/trans isomerases (PPIases) isomerise the peptide bond of specific phosphorylated (Ser/Thr)‐Pro residues, regulating various cellular events. Previously, we reported a Pin1‐type PPIase in Trypanosoma cruzi, but little is known about its function and subcellular localization. Immunofluorescence analysis revealed that in contrast with Pin1‐like proteins from diverse organisms, TcPin1 mainly localized in the cytoplasm and was excluded from the nuclei. In addition, RNAi‐mediated downregulation of TbPin1 in Trypanosoma brucei did not abolish cell proliferation. Using yeast two‐hybrid assay, we identified a MORN domain‐containing protein as putative Pin1‐binding partners. These data suggest that Pin1‐mediated signaling mechanism plays a different role in protozoan parasites.     

The Utilization of Glucose and Proline by Culture Forms of Trypanosoma brucei*

Exponentially dividing culture forms of Trypanosoma brucei did not utilize glucose provided in the culture medium. The inclusion of 2-deoxyglucose in the medium had no effect on the growth of the trypanosomes. Glucose could be replaced by proline in the liquid phase of biphasic medium without affecting the doubling time of the organisms. Proline added to the culture medium in this way disappeared during the log phase of growth. Glucose in the culture medium was used by the trypanosomes only when the stationary growth phase had been reached. Lipid accumulated in stationary phase trypanosomes grown in glucose-containing medium, but there was no lipid accumulation in log phase organisms or in those which had been grown in proline-containing medium. Bloodstream trypanosomes transferred to liquid medium rapidly utilized glucose over the first 12 hr of culture, and this was accompanied by an accumulation of free pyruvate in the medium. The rate of glucose utilization fell off over the next 36 hr; this was accompanied by a lowering of free pyruvate in the medium and a rise in the proline oxidase activity of the trypanosomes. The possible biologic significance of proline to trypanosomes developing in the midgut of the tsetse vector is discussed.     

Phosphoglucomutase is absent in Trypanosoma brucei and redundantly substituted by phosphomannomutase and phospho-N-acetylglucosamine mutase

The enzymes phosphomannomutase (PMM), phospho‐N‐acetylglucosamine mutase (PAGM) and phosphoglucomutase (PGM) reversibly catalyse the transfer of phosphate between the C6 and C1 hydroxyl groups of mannose, N‐acetylglucosamine and glucose respectively. Although genes for a candidate PMM and a PAGM enzymes have been found in the Trypanosoma brucei genome, there is, surprisingly, no candidate gene for PGM. The TbPMM and TbPAGM genes were cloned and expressed in Escherichia coli and the TbPMM enzyme was crystallized and its structure solved at 1.85 Å resolution. Antibodies to the recombinant proteins localized endogenous TbPMM to glycosomes in the bloodstream form of the parasite, while TbPAGM localized to both the cytosol and glycosomes. Both recombinant enzymes were able to interconvert glucose‐phosphates, as well as acting on their own definitive substrates. Analysis of sugar nucleotide levels in parasites with TbPMM or TbPAGM knocked down by RNA interference (RNAi) suggests that, in vivo, PGM activity is catalysed by both enzymes. This is the first example in any organism of PGM activity being completely replaced in this way and it explains why, uniquely, T. brucei has been able to lose its PGM gene. The RNAi data for TbPMM also showed that this is an essential gene for parasite growth.     

Forward motility is essential for trypanosome infection in the tsetse fly

African trypanosomes are flagellated protozoan parasites transmitted by the bite of tsetse flies and responsible for sleeping sickness in humans. Their complex development in the tsetse digestive tract requires several differentiation and migration steps that are thought to rely on trypanosome motility. We used a functional approach in vivo to demonstrate that motility impairment prevents trypanosomes from developing in their vector. Deletion of the outer dynein arm component DNAI1 results in strong motility defects but cells remain viable in culture. However, although these mutant trypanosomes could infect the tsetse fly midgut, they were neither able to reach the foregut nor able to differentiate into the next stage, thus failing to complete their parasite cycle. This is the first in vivo demonstration that trypanosome motility is essential for the accomplishment of the parasite cycle.     

Non-histone protein methylation in Trypanosoma cruzi epimastigotes

Post-translational methylation of proteins, which occurs in arginines and lysines, modulates several biological processes at different levels of cell signaling. Recently, methylation has been demonstrated in the regulation beyond histones, for example, in the dynamics of protein-protein and protein-nucleic acid interactions. However, the presence and role of non-histone methylation in Trypanosoma cruzi, the etiologic agent of Chagas disease, has not yet been elucidated. Here, we applied mass spectrometry-based-proteomics (LC-MS/MS) to profile the methylproteome of T. cruzi epimastigotes, describing a total of 1252 methyl sites in 824 proteins. Functional enrichment and protein-protein interaction analysis show that protein methylation impacts important biological processes of the parasite, such as translation, RNA and DNA binding, amino acid, and carbohydrate metabolism. In addition, 171 of the methylated proteins were previously reported to bear phosphorylation sites in T. cruzi, including flagellar proteins and RNA binding proteins, indicating that there may be an interplay between these different modifications in non-histone proteins. Our results show that a broad spectrum of functions is affected by methylation in T. cruzi, indicating its potential to impact important processes in the biology of the parasite and other trypanosomes.     

Tsetse EP Protein Protects the Fly Midgut from Trypanosome Establishment

African trypanosomes undergo a complex developmental process in their tsetse fly vector before transmission back to a vertebrate host. Typically, 90% of fly infections fail, most during initial establishment of the parasite in the fly midgut. The specific mechanism(s) underpinning this failure are unknown. We have previously shown that a Glossina-specific, immunoresponsive molecule, tsetse EP protein, is up regulated by the fly in response to gram-negative microbial challenge. Here we show by knockdown using RNA interference that this tsetse EP protein acts as a powerful antagonist of establishment in the fly midgut for both Trypanosoma brucei brucei and T. congolense. We demonstrate that this phenomenon exists in two species of tsetse, Glossina morsitans morsitans and G. palpalis palpalis, suggesting tsetse EP protein may be a major determinant of vector competence in all Glossina species. Tsetse EP protein levels also decline in response to starvation of the fly, providing a possible explanation for increased susceptibility of starved flies to trypanosome infection. As starvation is a common field event, this fact may be of considerable importance in the epidemiology of African trypanosomiasis.     

Systematic structural studies of iron superoxide dismutases from human parasites and a statistical coupling analysis of metal binding specificity

Superoxide dismutases (SODs) are a crucial class of enzymes in the combat against intracellular free radical damage. They eliminate superoxide radicals by converting them into hydrogen peroxide and oxygen. In spite of their very different life cycles and infection strategies, the human parasites Plasmodium falciparum, Trypanosoma cruzi and Trypanosoma brucei are known to be sensitive to oxidative stress. Thus the parasite Fe‐SODs have become attractive targets for novel drug development. Here we report the crystal structures of FeSODs from the trypanosomes T. brucei at 2.0 Å and T. cruzi at 1.9 Å resolution, and that from P. falciparum at a higher resolution (2.0 Å) to that previously reported. The homodimeric enzymes are compared to the related human MnSOD with particular attention to structural aspects which are relevant for drug design. Although the structures possess a very similar overall fold, differences between the enzymes at the entrance to the channel which leads to the active site could be identified. These lead to a slightly broader and more positively charged cavity in the parasite enzymes. Furthermore, a statistical coupling analysis (SCA) for the whole Fe/MnSOD family reveals different patterns of residue coupling for Mn and Fe SODs, as well as for the dimeric and tetrameric states. In both cases, the statistically coupled residues lie adjacent to the conserved core surrounding the metal center and may be expected to be responsible for its fine tuning, leading to metal ion specificity. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Knockdown of Asparagine Synthetase A Renders Trypanosoma brucei Auxotrophic to Asparagine

Asparagine synthetase (AS) catalyzes the ATP-dependent conversion of aspartate into asparagine using ammonia or glutamine as nitrogen source. There are two distinct types of AS, asparagine synthetase A (AS-A), known as strictly ammonia-dependent, and asparagine synthetase B (AS-B), which can use either ammonia or glutamine. The absence of AS-A in humans, and its presence in trypanosomes, suggested AS-A as a potential drug target that deserved further investigation. We report the presence of functional AS-A in Trypanosoma cruzi (TcAS-A) and Trypanosoma brucei (TbAS-A): the purified enzymes convert L-aspartate into L-asparagine in the presence of ATP, ammonia and Mg²⁺. TcAS-A and TbAS-A use preferentially ammonia as a nitrogen donor, but surprisingly, can also use glutamine, a characteristic so far never described for any AS-A. TbAS-A knockdown by RNAi didn''t affect in vitro growth of bloodstream forms of the parasite. However, growth was significantly impaired when TbAS-A knockdown parasites were cultured in medium with reduced levels of asparagine. As expected, mice infections with induced and non-induced T. brucei RNAi clones were similar to those from wild-type parasites. However, when induced T. brucei RNAi clones were injected in mice undergoing asparaginase treatment, which depletes blood asparagine, the mice exhibited lower parasitemia and a prolonged survival in comparison to similarly-treated mice infected with control parasites. Our results show that TbAS-A can be important under in vivo conditions when asparagine is limiting, but is unlikely to be suitable as a drug target.     

Cell cycle regulation and novel structural features of thymidine kinase,an essential enzyme in Trypanosoma brucei

Thymidine kinase (TK) is a key enzyme in the pyrimidine salvage pathway which catalyzes the transfer of the γ‐phosphate of ATP to 2′‐deoxythymidine (dThd) forming thymidine monophosphate (dTMP). Unlike other type II TKs, the Trypanosoma brucei enzyme (TbTK) is a tandem protein with two TK homolog domains of which only the C‐terminal one is active. In this study, we establish that TbTK is essential for parasite viability and cell cycle progression, independently of extracellular pyrimidine concentrations. We show that expression of TbTK is cell cycle regulated and that depletion of TbTK leads to strongly diminished dTTP pools and DNA damage indicating intracellular dThd to be an essential intermediate metabolite for the synthesis of thymine‐derived nucleotides. In addition, we report the X‐ray structure of the catalytically active domain of TbTK in complex with dThd and dTMP at resolutions up to 2.2 Å. In spite of the high conservation of the active site residues, the structures reveal a widened active site cavity near the nucleobase moiety compared to the human enzyme. Our findings strongly support TbTK as a crucial enzyme in dTTP homeostasis and identify structural differences within the active site that could be exploited in the process of rational drug design.     

Antigenic Analysis By Immunofluorescence of In Vitro-Produced Metacyclics of Trypanosoma Brucei and Their Infections In Mice*

SYNOPSIS. the antigenic types in populations of metacyclic trypanosomes of Trypanosoma brucei isolated from Glossina morsitans head-salivary gland trypanosome cultures and bloodstream forms in the early parasitemias produced from whole culture supernatant fluids containing metacyclic forms, were analyzed by the indirect fluorescent antibody test using clone-specific antisera. Metacyclic trypanosomes in cultures initiated with cloned bloodstream forms were heterogeneous with respect to their variable antigenic type (VAT). Trypanosomes comprising early parasitemias in immunosuppressed mice infected with metacyclics produced in cultures also had a range of VATs. Three of the VATs detected in the early parasitemias in mice have also been identified by other investigators in tsetse fly-transmitted populations of the same stock.