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1.  Biochemical characterization of the initial steps of the Kennedy pathway in Trypanosoma brucei: the ethanolamine and choline kinases 
Biochemical Journal  2008;415(Pt 1):135-144.
Ethanolamine and choline are major components of the trypanosome membrane phospholipids, in the form of GPEtn (glycerophosphoethanolamine) and GPCho (glycerophosphocholine). Ethanolamine is also found as an integral component of the GPI (glycosylphosphatidylinositol) anchor that is required for membrane attachment of cell-surface proteins, most notably the variant-surface glycoproteins. The de novo synthesis of GPEtn and GPCho starts with the generation of phosphoethanolamine and phosphocholine by ethanolamine and choline kinases via the Kennedy pathway. Database mining revealed two putative C/EKs (choline/ethanolamine kinases) in the Trypanosoma brucei genome, which were cloned, overexpressed, purified and characterized. TbEK1 (T. brucei ethanolamine kinase 1) was shown to be catalytically active as an ethanolamine-specific kinase, i.e. it had no choline kinase activity. The Km values for ethanolamine and ATP were found to be 18.4±0.9 and 219±29 μM respectively. TbC/EK2 (T. brucei choline/ethanolamine kinase 2), on the other hand, was found to be able to phosphorylate both ethanolamine and choline, even though choline was the preferred substrate, with a Km 80 times lower than that of ethanolamine. The Km values for choline, ethanolamine and ATP were 31.4±2.6 μM, 2.56±0.31 mM and 20.6±1.96 μM respectively. Further substrate specificity analysis revealed that both TbEK1 and TbC/EK2 were able to tolerate various modifications at the amino group, with the exception of a quaternary amine for TbEK1 (choline) and a primary amine for TbC/EK2 (ethanolamine). Both enzymes recognized analogues with substituents on C-2, but substitutions on C-1 and elongations of the carbon chain were not well tolerated.
PMCID: PMC2552378  PMID: 18489261
choline kinase; ethanolamine kinase; Kennedy pathway; Trypanosoma brucei; C/EK, choline/ethanolamine kinase; EK, ethanolamine kinase; GPCho, glycerophosphocholine; GPEtn, glycerophosphoethanolamine; GPI, glycosylphosphatidylinositol; GPSer, glycerophosphoserine; HPTLC, high-performance TLC; LB, Luria–Bertani; MALDI, matrix-assisted laser-desorption ionization; ORF, open reading frame; PtdCho, phosphotidylcholine; PtdEtn, phosphatidylethanolamine; RT, reverse transcription; Tb, Trypanosome brucei; TEV, tobacco etch virus; TOF, time-of-flight; UTR, untranslated region; VSG, variant-surface glycoprotein
2.  Kinetic modelling of phospholipid synthesis in Plasmodium knowlesi unravels crucial steps and relative importance of multiple pathways 
BMC Systems Biology  2013;7:123.
Plasmodium is the causal parasite of malaria, infectious disease responsible for the death of up to one million people each year. Glycerophospholipid and consequently membrane biosynthesis are essential for the survival of the parasite and are targeted by a new class of antimalarial drugs developed in our lab. In order to understand the highly redundant phospholipid synthethic pathways and eventual mechanism of resistance to various drugs, an organism specific kinetic model of these metabolic pathways need to be developed in Plasmodium species.
Fluxomic data were used to build a quantitative kinetic model of glycerophospholipid pathways in Plasmodium knowlesi. In vitro incorporation dynamics of phospholipids unravels multiple synthetic pathways. A detailed metabolic network with values of the kinetic parameters (maximum rates and Michaelis constants) has been built. In order to obtain a global search in the parameter space, we have designed a hybrid, discrete and continuous, optimization method. Discrete parameters were used to sample the cone of admissible fluxes, whereas the continuous Michaelis and maximum rates constants were obtained by local minimization of an objective function.The model was used to predict the distribution of fluxes within the network of various metabolic precursors.
The quantitative analysis was used to understand eventual links between different pathways. The major source of phosphatidylcholine (PC) is the CDP-choline Kennedy pathway.
In silico knock-out experiments showed comparable importance of phosphoethanolamine-N-methyltransferase (PMT) and phosphatidylethanolamine-N-methyltransferase (PEMT) for PC synthesis.
The flux values indicate that, major part of serine derived phosphatidylethanolamine (PE) is formed via serine decarboxylation, whereas major part of phosphatidylserine (PS) is formed by base-exchange reactions.
Sensitivity analysis of CDP-choline pathway shows that the carrier-mediated choline entry into the parasite and the phosphocholine cytidylyltransferase reaction have the largest sensitivity coefficients in this pathway, but does not distinguish a reaction as an unique rate-limiting step.
We provide a fully parametrized kinetic model for the multiple phospholipid synthetic pathways in P. knowlesi. This model has been used to clarify the relative importance of the various reactions in these metabolic pathways. Future work extensions of this modelling strategy will serve to elucidate the regulatory mechanisms governing the development of Plasmodium during its blood stages, as well as the mechanisms of action of drugs on membrane biosynthetic pathways and eventual mechanisms of resistance.
PMCID: PMC3829661  PMID: 24209716
Malaria; Phospholipid metabolism; Plasmodium knowlesi; Mathematical model; Fluxomics; Hybrid optimization
3.  T. brucei Infection Reduces B Lymphopoiesis in Bone Marrow and Truncates Compensatory Splenic Lymphopoiesis through Transitional B-Cell Apoptosis 
PLoS Pathogens  2011;7(6):e1002089.
African trypanosomes of the Trypanosoma brucei species are extracellular protozoan parasites that cause the deadly disease African trypanosomiasis in humans and contribute to the animal counterpart, Nagana. Trypanosome clearance from the bloodstream is mediated by antibodies specific for their Variant Surface Glycoprotein (VSG) coat antigens. However, T. brucei infection induces polyclonal B cell activation, B cell clonal exhaustion, sustained depletion of mature splenic Marginal Zone B (MZB) and Follicular B (FoB) cells, and destruction of the B-cell memory compartment. To determine how trypanosome infection compromises the humoral immune defense system we used a C57BL/6 T. brucei AnTat 1.1 mouse model and multicolor flow cytometry to document B cell development and maturation during infection. Our results show a more than 95% reduction in B cell precursor numbers from the CLP, pre-pro-B, pro-B, pre-B and immature B cell stages in the bone marrow. In the spleen, T. brucei induces extramedullary B lymphopoiesis as evidenced by significant increases in HSC-LMPP, CLP, pre-pro-B, pro-B and pre-B cell populations. However, final B cell maturation is abrogated by infection-induced apoptosis of transitional B cells of both the T1 and T2 populations which is not uniquely dependent on TNF-, Fas-, or prostaglandin-dependent death pathways. Results obtained from ex vivo co-cultures of living bloodstream form trypanosomes and splenocytes demonstrate that trypanosome surface coat-dependent contact with T1/2 B cells triggers their deletion. We conclude that infection-induced and possibly parasite-contact dependent deletion of transitional B cells prevents replenishment of mature B cell compartments during infection thus contributing to a loss of the host's capacity to sustain antibody responses against recurring parasitemic waves.
Author Summary
African trypanosomiasis caused by Trypanosoma brucei species is fatal in both humans and animals and cannot be combated by vaccination because of extensive parasite antigenic variation. Effective trypanosome control and clearance from the bloodstream involves the action of antibodies specific for the parasite's highly diverse variable surface glycoprotein antigens. However, experimental infections in mice have shown that trypanosomiasis elicits a rapid process of B cell exhaustion and loss of protective antibody responses. Indeed, both marginal zone B cells, the first line of defense against blood-borne pathogens like T. brucei parasites, and follicular B cells, which are the major source for developing high-affinity antibody-producing plasma cells and memory B cells, become depleted during infection. In addition, existing B-cell memory, both against parasite antigens and non related pathogens, is destroyed early on in infection. Here, we demonstrate that during infection, B cell development is decreased in the bone marrow and early B cell development is taken over by the spleen. However, full maturation of developing B cells is abrogated by the occurrence of transitional B cell apoptosis. This impairs the replenishment of the mature marginal zone and follicular B cell pools and prevents the buildup of protective immunity against successive parasitemic waves.
PMCID: PMC3128123  PMID: 21738467
4.  Cytosolic Peroxidases Protect the Lysosome of Bloodstream African Trypanosomes from Iron-Mediated Membrane Damage 
PLoS Pathogens  2014;10(4):e1004075.
African trypanosomes express three virtually identical non-selenium glutathione peroxidase (Px)-type enzymes which preferably detoxify lipid-derived hydroperoxides. As shown previously, bloodstream Trypanosoma brucei lacking the mitochondrial Px III display only a weak and transient proliferation defect whereas parasites that lack the cytosolic Px I and Px II undergo extremely fast lipid peroxidation and cell lysis. The phenotype can completely be rescued by supplementing the medium with the α-tocopherol derivative Trolox. The mechanism underlying the rapid cell death remained however elusive. Here we show that the lysosome is the origin of the cellular injury. Feeding the px I–II knockout parasites with Alexa Fluor-conjugated dextran or LysoTracker in the presence of Trolox yielded a discrete lysosomal staining. Yet upon withdrawal of the antioxidant, the signal became progressively spread over the whole cell body and was completely lost, respectively. T. brucei acquire iron by endocytosis of host transferrin. Supplementing the medium with iron or transferrin induced, whereas the iron chelator deferoxamine and apo-transferrin attenuated lysis of the px I–II knockout cells. Immunofluorescence microscopy with MitoTracker and antibodies against the lysosomal marker protein p67 revealed that disintegration of the lysosome precedes mitochondrial damage. In vivo experiments confirmed the negligible role of the mitochondrial peroxidase: Mice infected with px III knockout cells displayed only a slightly delayed disease development compared to wild-type parasites. Our data demonstrate that in bloodstream African trypanosomes, the lysosome, not the mitochondrion, is the primary site of oxidative damage and cytosolic trypanothione/tryparedoxin-dependent peroxidases protect the lysosome from iron-induced membrane peroxidation. This process appears to be closely linked to the high endocytic rate and distinct iron acquisition mechanisms of the infective stage of T. brucei. The respective knockout of the cytosolic px I–II in the procyclic insect form resulted in cells that were fully viable in Trolox-free medium.
Author Summary
In many cell types, mitochondria are the main source of intracellular reactive oxygen species but iron-induced oxidative lysosomal damage has been described as well. African trypanosomes are the causative agents of human sleeping sickness and the cattle disease Nagana. The parasites are obligate extracellular pathogens that multiply in the bloodstream and body fluids of their mammalian hosts and as procyclic forms in their insect vector, the tsetse fly. Bloodstream Trypanosoma brucei in which the genes for cytosolic lipid hydroperoxide-detoxifying peroxidases have been knocked out undergo an extremely rapid membrane peroxidation and lyse within less than two hours when they are cultured without an exogenous antioxidant. Here we show that the primary site of intracellular damage is the single terminal lysosome of the parasites. Disintegration of the lysosome clearly precedes damage of the mitochondrion and parasite death. Iron, acquired by the endocytosis of iron-loaded host transferrin, induces cell lysis. Contrary to the cytosolic enzymes, the respective mitochondrial peroxidase is dispensable for both in vitro proliferation and mouse infectivity. This is the first report demonstrating that cytosolic thiol peroxidases are responsible for protecting the lysosome of a cell.
PMCID: PMC3983053  PMID: 24722489
5.  The life cycle of Trypanosoma (Nannomonas) congolense in the tsetse fly 
Parasites & Vectors  2012;5:109.
The tsetse-transmitted African trypanosomes cause diseases of importance to the health of both humans and livestock. The life cycles of these trypanosomes in the fly were described in the last century, but comparatively few details are available for Trypanosoma (Nannomonas) congolense, despite the fact that it is probably the most prevalent and widespread pathogenic species for livestock in tropical Africa. When the fly takes up bloodstream form trypanosomes, the initial establishment of midgut infection and invasion of the proventriculus is much the same in T. congolense and T. brucei. However, the developmental pathways subsequently diverge, with production of infective metacyclics in the proboscis for T. congolense and in the salivary glands for T. brucei. Whereas events during migration from the proventriculus are understood for T. brucei, knowledge of the corresponding developmental pathway in T. congolense is rudimentary. The recent publication of the genome sequence makes it timely to re-investigate the life cycle of T. congolense.
Experimental tsetse flies were fed an initial bloodmeal containing T. congolense strain 1/148 and dissected 2 to 78 days later. Trypanosomes recovered from the midgut, proventriculus, proboscis and cibarium were fixed and stained for digital image analysis. Trypanosomes contained in spit samples from individually caged flies were analysed similarly. Mensural data from individual trypanosomes were subjected to principal components analysis.
Flies were more susceptible to infection with T. congolense than T. brucei; a high proportion of flies infected with T. congolense established a midgut and subsequent proboscis infection, whereas many T. brucei infections were lost in the migration from foregut to salivary glands. In T. congolense, trypomastigotes ceased division in the proventriculus and became uniform in size. The trypanosomes retained trypomastigote morphology during migration via the foregut to the mouthparts and we confirmed that the trypomastigote-epimastigote transition occurred in the proboscis. We found no equivalent to the asymmetric division stage in T. brucei that mediates transition of proventricular trypomastigotes to epimastigotes. In T. congolense extremely long epimastigotes with remarkably elongated posterior ends were observed in both the proboscis and cibarium; no difference was found in the developmental stages in these two organs. Dividing trypomastigotes and epimastigotes were recovered from the proboscis, some of which were in transition from trypomastigote to epimastigote and vice versa. It remains uncertain whether these morphological transitions are mediated by cell division, since we also found non-dividing cells with a variously positioned, juxta-nuclear kinetoplast.
We have presented a detailed description of the life cycle of T. congolense in its tsetse fly vector. During development in the fly T. congolense shares a common migratory pathway with its close relative T. brucei, culminating in the production of small metacyclic trypanosomes that can be inoculated with the saliva. Despite this outward similarity in life cycle, the transitional developmental stages in the foregut and mouthparts are remarkably different in the two trypanosome species.
PMCID: PMC3384477  PMID: 22676292
6.  Regulation of the Saccharomyces cerevisiae EKI1-encoded Ethanolamine Kinase by Zinc Depletion* 
The Journal of biological chemistry  2006;281(19):13110-13116.
Ethanolamine kinase catalyzes the committed step in the synthesis of phosphatidylethanolamine via the CDP-ethanolamine branch of the Kennedy pathway. Regulation of the EKI1-encoded ethanolamine kinase by the essential nutrient zinc was examined in Saccharomyces cerevisiae. The level of ethanolamine kinase activity increased when zinc was depleted from the growth medium. This regulation correlated with increases in the CDP-ethanolamine pathway intermediates phosphoethanolamine and CDP-ethanolamine, and an increase in the methylated derivative of phosphatidylethanolamine, phosphatidylcholine. The β-galactosidase activity driven by the PEKI1-lacZ reporter gene was elevated in zinc-depleted cells, indicating that the increase in ethanolamine kinase activity was attributed to a transcriptional mechanism. The expression level of PEKI1-lacZ reporter gene activity in the zrt1Δzrt2Δ mutant (defective in plasma membrane zinc transport) cells grown with zinc was similar to the activity expressed in wild-type cells grown without zinc. This indicated that EKI1 expression was sensitive to intracellular zinc. The zinc-mediated regulation of EKI1 expression was attenuated in the zap1Δ mutant defective in the zinc-regulated transcription factor Zap1p. Direct interactions between Zap1p and putative zinc-responsive elements in the EKI1 promoter were demonstrated by electrophoretic mobility shift assays. Mutations of these elements to a nonconsensus sequence abolished Zap1p-DNA interactions. Taken together, this work demonstrated that the zinc-mediated regulation of ethanolamine kinase and the synthesis of phospholipids via the CDP-ethanolamine branch of the Kennedy pathway were controlled in part by Zap1p.
PMCID: PMC1779367  PMID: 16551612
7.  The essential neutral sphingomyelinase is involved in the trafficking of the variant surface glycoprotein in the bloodstream form of Trypanosoma brucei 
Molecular Microbiology  2010;76(6):1461-1482.
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.
PMCID: PMC2904498  PMID: 20398210
8.  CTP synthetase and its role in phospholipid synthesis in the yeast Saccharomyces cerevisiae 
Progress in lipid research  2008;47(5):333-339.
CTP synthetase is a cytosolic-associated glutamine amidotransferase enzyme that catalyzes the ATP-dependent transfer of the amide nitrogen from glutamine to the C-4 position of UTP to form CTP. In the yeast Saccharomyces cerevisiae, the reaction product CTP is an essential precursor of all membrane phospholipids that are synthesized via the Kennedy (CDP-choline and CDP-ethanolamine branches) and CDP-diacylglycerol pathways. The URA7 and URA8 genes encode CTP synthetase in S. cerevisiae, and the URA7 gene is responsible for the majority of CTP synthesized in vivo. The CTP synthetase enzymes are allosterically regulated by CTP product inhibition. Mutations that alleviate this regulation result in an elevated cellular level of CTP and an increase in phospholipid synthesis via the Kennedy pathway. The URA7-encoded enzyme is phosphorylated by protein kinases A and C, and these phosphorylations stimulate CTP synthetase activity and increase cellular CTP levels and the utilization of the Kennedy pathway. The CTPS1 and CTPS2 genes that encode human CTP synthetase enzymes are functionally expressed in S. cerevisiae, and rescue the lethal phenotype of the ura7Δ ura8Δ double mutant that lacks CTP synthetase activity. The expression in yeast has revealed that the human CTPS1-encoded enzyme is also phosphorylated and regulated by protein kinases A and C.
PMCID: PMC2583782  PMID: 18439916
CTP; CTP synthetase; CDP-diacylglycerol; CDP-choline; CDP-ethanolamine; phospholipid synthesis; phosphorylation
9.  A Cell-surface Phylome for African Trypanosomes 
The cell surface of Trypanosoma brucei, like many protistan blood parasites, is crucial for mediating host-parasite interactions and is instrumental to the initiation, maintenance and severity of infection. Previous comparisons with the related trypanosomatid parasites T. cruzi and Leishmania major suggest that the cell-surface proteome of T. brucei is largely taxon-specific. Here we compare genes predicted to encode cell surface proteins of T. brucei with those from two related African trypanosomes, T. congolense and T. vivax. We created a cell surface phylome (CSP) by estimating phylogenies for 79 gene families with putative surface functions to understand the more recent evolution of African trypanosome surface architecture. Our findings demonstrate that the transferrin receptor genes essential for bloodstream survival in T. brucei are conserved in T. congolense but absent from T. vivax and include an expanded gene family of insect stage-specific surface glycoproteins that includes many currently uncharacterized genes. We also identify species-specific features and innovations and confirm that these include most expression site-associated genes (ESAGs) in T. brucei, which are absent from T. congolense and T. vivax. The CSP presents the first global picture of the origins and dynamics of cell surface architecture in African trypanosomes, representing the principal differences in genomic repertoire between African trypanosome species and provides a basis from which to explore the developmental and pathological differences in surface architectures. All data can be accessed at:
Author Summary
The African trypanosome (Trypanosoma brucei) is a single-celled, vector-borne parasite that causes Human African Trypanosomiasis (or ‘sleeping sickness’) throughout sub-Saharan Africa and, along with related species T. congolense and T. vivax, a similar disease in wild and domestic animals. Together, the African trypanosomes have significant effects on human and animal health and associated costs for socio-economic development in Africa. Genes expressed on the trypanosome cell surface are instrumental in causing disease and sustaining infection by resisting the host immune system. Here we compare repertoires of genes with predicted cell-surface expression in T. brucei, T. congolense and T. vivax and estimate the phylogeny of each predicted cell-surface gene family. This ‘cell-surface phylome’ (CSP) provides a detailed analysis of species-specific gene families and of gene gain and loss in shared families, aiding the identification of surface proteins that may mediate specific aspects of pathogenesis and disease progression. Overall, the CSP suggests that each trypanosome species has modified its surface proteome uniquely, indicating that T. brucei, T. congolense and T. vivax have subtly distinct mechanisms for interacting with both vertebrate and insect hosts.
PMCID: PMC3605285  PMID: 23556014
10.  Revisiting the Central Metabolism of the Bloodstream Forms of Trypanosoma brucei: Production of Acetate in the Mitochondrion Is Essential for Parasite Viability 
The bloodstream forms of Trypanosoma brucei, the causative agent of sleeping sickness, rely solely on glycolysis for ATP production. It is generally accepted that pyruvate is the major end-product excreted from glucose metabolism by the proliferative long-slender bloodstream forms of the parasite, with virtually no production of succinate and acetate, the main end-products excreted from glycolysis by all the other trypanosomatid adaptative forms, including the procyclic insect form of T. brucei.
Methodology/Principal Findings
A comparative NMR analysis showed that the bloodstream long-slender and procyclic trypanosomes excreted equivalent amounts of acetate and succinate from glucose metabolism. Key enzymes of acetate production from glucose-derived pyruvate and threonine are expressed in the mitochondrion of the long-slender forms, which produces 1.4-times more acetate from glucose than from threonine in the presence of an equal amount of both carbon sources. By using a combination of reverse genetics and NMR analyses, we showed that mitochondrial production of acetate is essential for the long-slender forms, since blocking of acetate biosynthesis from both carbon sources induces cell death. This was confirmed in the absence of threonine by the lethal phenotype of RNAi-mediated depletion of the pyruvate dehydrogenase, which is involved in glucose-derived acetate production. In addition, we showed that de novo fatty acid biosynthesis from acetate is essential for this parasite, as demonstrated by a lethal phenotype and metabolic analyses of RNAi-mediated depletion of acetyl-CoA synthetase, catalyzing the first cytosolic step of this pathway.
Acetate produced in the mitochondrion from glucose and threonine is synthetically essential for the long-slender mammalian forms of T. brucei to feed the essential fatty acid biosynthesis through the “acetate shuttle” that was recently described in the procyclic insect form of the parasite. Consequently, key enzymatic steps of this pathway, particularly acetyl-CoA synthetase, constitute new attractive drug targets against trypanosomiasis.
Author Summary
Many protists, including parasitic helminthes, trichomonads and trypanosomatids, produce acetate in their mitochondrion or mitochondrion-like organelle, which is excreted as a main metabolic end-product of their energy metabolism. We have recently demonstrated that mitochondrial production of acetate is essential for fatty acid biosynthesis and ATP production in the procyclic insect form of T. brucei. However, acetate metabolism has not been investigated in the long-slender bloodstream forms of the parasite, the proliferative forms responsible for the sleeping sickness. In contrast to the current view, we showed that the bloodstream forms produce almost as much acetate from glucose than the procyclic parasites. Acetate production from glucose and threonine is synthetically essential for growth and de novo synthesis of fatty acids of the bloodstream trypanosomes. These data highlight that the central metabolism of the bloodstream forms contains unexpected essential pathways, although minor in terms of metabolic flux, which could be targeted for the development of trypanocidal drugs.
PMCID: PMC3868518  PMID: 24367711
11.  Regulators of Trypanosoma brucei Cell Cycle Progression and Differentiation Identified Using a Kinome-Wide RNAi Screen 
PLoS Pathogens  2014;10(1):e1003886.
The African trypanosome, Trypanosoma brucei, maintains an integral link between cell cycle regulation and differentiation during its intricate life cycle. Whilst extensive changes in phosphorylation have been documented between the mammalian bloodstream form and the insect procyclic form, relatively little is known about the parasite's protein kinases (PKs) involved in the control of cellular proliferation and differentiation. To address this, a T. brucei kinome-wide RNAi cell line library was generated, allowing independent inducible knockdown of each of the parasite's 190 predicted protein kinases. Screening of this library using a cell viability assay identified ≥42 PKs that are required for normal bloodstream form proliferation in culture. A secondary screen identified 24 PKs whose RNAi-mediated depletion resulted in a variety of cell cycle defects including in G1/S, kinetoplast replication/segregation, mitosis and cytokinesis, 15 of which are novel cell cycle regulators. A further screen identified for the first time two PKs, named repressor of differentiation kinase (RDK1 and RDK2), depletion of which promoted bloodstream to procyclic form differentiation. RDK1 is a membrane-associated STE11-like PK, whilst RDK2 is a NEK PK that is essential for parasite proliferation. RDK1 acts in conjunction with the PTP1/PIP39 phosphatase cascade to block uncontrolled bloodstream to procyclic form differentiation, whilst RDK2 is a PK whose depletion efficiently induces differentiation in the absence of known triggers. Thus, the RNAi kinome library provides a valuable asset for functional analysis of cell signalling pathways in African trypanosomes as well as drug target identification and validation.
Author Summary
The African trypanosome, which is transmitted by the tsetse fly, causes the usually fatal disease Sleeping Sickness in humans and a wasting disease, called Nagana, in livestock in sub-Saharan Africa. There are no vaccines available against the diseases, and various problems are associated with current drug treatments (including toxicity to the patient and parasite drug resistance). Thus, it is important to identify essential parasite proteins that could be targeted by novel drugs. Protein kinases (PKs) are important cell signalling molecules, and are generally considered to have potential as drug targets. Here we report the construction of a library of trypanosome cell lines that allows us to specifically deplete each of the trypanosome's 190 PKs individually and analyse their function. Using this library, we show that ≥42 PKs are essential for proliferation of the mammalian-infective bloodstream form of the parasite (and thus have potential as drug targets), and demonstrate that 24 of these play important roles in coordinating cell division. We also shed light on how the parasite develops during its life cycle as it passes from the mammalian bloodstream form to the tsetse fly gut by identifying the first two PKs that regulate this life cycle developmental step.
PMCID: PMC3894213  PMID: 24453978
12.  Identification of Trans-Sialidases as a Common Mediator of Endothelial Cell Activation by African Trypanosomes 
PLoS Pathogens  2013;9(10):e1003710.
Understanding African Trypanosomiasis (AT) host-pathogen interaction is the key to an “anti-disease vaccine”, a novel strategy to control AT. Here we provide a better insight into this poorly described interaction by characterizing the activation of a panel of endothelial cells by bloodstream forms of four African trypanosome species, known to interact with host endothelium. T. congolense, T. vivax, and T. b. gambiense activated the endothelial NF-κB pathway, but interestingly, not T. b. brucei. The parasitic TS (trans-sialidases) mediated this NF-κB activation, remarkably via their lectin-like domain and induced production of pro-inflammatory molecules not only in vitro but also in vivo, suggesting a considerable impact on pathogenesis. For the first time, TS activity was identified in T. b. gambiense BSF which distinguishes it from the subspecies T. b. brucei. The corresponding TS were characterized and shown to activate endothelial cells, suggesting that TS represent a common mediator of endothelium activation among trypanosome species with divergent physiopathologies.
Author Summary
African trypanosomiasis remains by far the most devastating parasitic disease in Africa affecting both humans and livestock. The current control strategies are not efficient because of the increasing resistance to trypanocidal drugs, and the antigenic variation that impedes vaccine development. An alternative strategy aiming to neutralize the pathological effects of the parasite rather than eliminate it was proposed. Therefore, it is essential to understand the development of pathogenesis and characterize the pathogenic factors. In this context, we wanted to elucidate the host-pathogen interaction between the African trypanosomes and the mammalian host endothelium. For the first time, we clearly demonstrated that animal African trypanosomes activate the endothelial cells via the NF-κB pathway and cause a pro-inflammatory response in vitro and in vivo via their TS. By comparing four different trypanosomes species, we showed that they displayed distinct capacities for activation. For the first time, we identified sialidase activity in the human parasite T. brucei gambiense and showed that sialidases are the mediators of this endothelial activation, in both human and animal trypanosomes. Interestingly, the lectin-like domain of this enzyme was responsible for the activation rather than the catalytic site. This study brings considerable insights into the host-pathogen relationship and designates the sialidases as a perfect target for an anti-disease strategy.
PMCID: PMC3795030  PMID: 24130501
13.  Identification of Compounds with Anti-Proliferative Activity against Trypanosoma brucei brucei Strain 427 by a Whole Cell Viability Based HTS Campaign 
Human African Trypanosomiasis (HAT) is caused by two trypanosome sub-species, Trypanosoma brucei rhodesiense and Trypanosoma brucei gambiense. Drugs available for the treatment of HAT have significant issues related to difficult administration regimes and limited efficacy across species and disease stages. Hence, there is considerable need to find new alternative and less toxic drugs. An approach to identify starting points for new drug candidates is high throughput screening (HTS) of large compound library collections. We describe the application of an Alamar Blue based, 384-well HTS assay to screen a library of 87,296 compounds against the related trypanosome subspecies, Trypanosoma brucei brucei bloodstream form lister 427. Primary hits identified against T.b. brucei were retested and the IC50 value compounds were estimated for T.b. brucei and a mammalian cell line HEK293, to determine a selectivity index for each compound. The screening campaign identified 205 compounds with greater than 10 times selectivity against T.b. brucei. Cluster analysis of these compounds, taking into account chemical and structural properties required for drug-like compounds, afforded a panel of eight compounds for further biological analysis. These compounds had IC50 values ranging from 0.22 µM to 4 µM with associated selectivity indices ranging from 19 to greater than 345. Further testing against T.b. rhodesiense led to the selection of 6 compounds from 5 new chemical classes with activity against the causative species of HAT, which can be considered potential candidates for HAT early drug discovery. Structure activity relationship (SAR) mining revealed components of those hit compound structures that may be important for biological activity. Four of these compounds have undergone further testing to 1) determine whether they are cidal or static in vitro at the minimum inhibitory concentration (MIC), and 2) estimate the time to kill.
Author Summary
Human African Sleeping Sickness (HAT) is a disease caused by sub-species of Trypanosoma. The disease affects developing countries within Africa, mainly occurring in rural regions that lack resources to purchase drugs for treatment. Drugs that are currently available have significant side effects, and treatment regimes are lengthy and not always transferrable to the field. In consideration of these factors, new drugs are urgently needed for the treatment of HAT. To discover compounds suitable for drug discovery, cultured trypanosomes can be tested against libraries of compounds to identify candidates for further biological analysis. We have utilised a 384-well format, Alamar Blue viability assay to screen a large non-proprietary compound collection against Trypanosoma brucei brucei bloodstream form lister 427. The assay was shown to be reproducible, with reference compounds exhibiting activity in agreement with previously published results. Primary screening hits were retested against T.b. brucei and HEK293 mammalian cells in order to assess selectivity against the parasite. Selective hits were characterised by chemical analysis, taking into consideration drug-like properties amenable to further progression. Priority compounds were tested against a panel of protozoan parasites, including Trypanosoma brucei rhodesiense, Trypanosoma cruzi, Leishmania donovani and Plasmodium falciparum. Five new compound classes were discovered that are amenable to progression in the drug discovery process for HAT.
PMCID: PMC3510080  PMID: 23209849
14.  Subcellular localization of a variable surface glycoprotein phosphatidylinositol-specific phospholipase-C in African trypanosomes 
The Journal of Cell Biology  1987;105(2):737-746.
African trypanosomes contain a membrane-bound enzyme capable of removing dimyristylglycerol from the membrane-attached form of the variable surface glycoprotein (mfVSG; Ferguson, M. A. J., K. Halder, and G. A. M. Cross, 1985, J. Biol Chem., 260:4963-4968). Although mfVSG phospholipase-C has been implicated in the removal of the VSG from the trypanosome surface (Cardoso de Almeida, M. L., and M. J. Turner, 1983, Nature (Lond.)., 302:349-352; Ferguson, M. A. J., K. Halder, and G. A. M. Cross, 1985, J. Biol Chem., 260:4963-4968), its precise function and subcellular location have not been determined. We have developed a procedure for the separation of the cell fractions and organelles of Trypanosoma brucei brucei (and other trypanosome species) by differential sucrose and isopycnic PercollR centrifugation. These fractions were tested for mfVSG phospholipase activity using Trypanosoma brucei mfVSG labeled with 3H-myristic acid as substrate. The highest enzyme-specific activity was associated with the flagella and evidence is presented to suggest that it is localized in the flagellar pocket. Some activity was also associated with the Golgi complex. These results suggest that the mfVSG phospholipase is localized primarily in the membrane of the flagella pocket and possibly other membrane organelles derived from and associated with this structure, and may be part of the VSG-membrane recycling system in African trypanosomes. The activity of mfVSG phospholipase amongst various trypanosome species was determined. We show that, in contrast to the bloodstream forms of Trypanosoma brucei, cultured procyclic Trypanosoma brucei and bloodstream Trypanosoma vivax had little or no mfVSG phospholipase activity. The activity found in bloodstream forms of Trypanosoma congolense was intermediate between Trypanosoma vivax and Trypanosoma brucei.
PMCID: PMC2114756  PMID: 3624307
15.  Regulated Expression of an Essential Allosteric Activator of Polyamine Biosynthesis in African Trypanosomes 
PLoS Pathogens  2008;4(10):e1000183.
Trypanosoma brucei is the causative agent of African sleeping sickness. The polyamine biosynthetic pathway has the distinction of being the target of the only clinically proven anti-trypanosomal drug with a known mechanism of action. Polyamines are essential for cell growth, and their metabolism is extensively regulated. However, trypanosomatids appear to lack the regulatory control mechanisms described in other eukaryotic cells. In T. brucei, S-adenosylmethionine decarboxylase (AdoMetDC) and ornithine decarboxylase (ODC) are required for the synthesis of polyamines and also for the unique redox-cofactor trypanothione. Further, trypanosomatid AdoMetDC is activated by heterodimer formation with a catalytically dead homolog termed prozyme, found only in these species. To study polyamine regulation in T. brucei, we generated inducible AdoMetDC RNAi and prozyme conditional knockouts in the mammalian blood form stage. Depletion of either protein led to a reduction in spermidine and trypanothione and to parasite death, demonstrating that prozyme activation of AdoMetDC is essential. Under typical growth conditions, prozyme concentration is limiting in comparison to AdoMetDC. However, both prozyme and ODC protein levels were significantly increased relative to stable transcript levels by knockdown of AdoMetDC or its chemical inhibition. Changes in protein stability do not appear to account for the increased steady-state protein levels, as both enzymes are stable in the presence of cycloheximide. These observations suggest that prozyme and ODC are translationally regulated in response to perturbations in the pathway. In conclusion, we describe the first evidence for regulation of polyamine biosynthesis in T. brucei and we demonstrate that the unique regulatory subunit of AdoMetDC is a key component of this regulation. The data support ODC and AdoMetDC as the key control points in the pathway and the likely rate-limiting steps in polyamine biosynthesis.
Author Summary
Human African trypanosomiasis (HAT) is an important vector-borne pathogen. The World Health Organization estimates that more than 50 million people are at risk for the disease, which occurs focally, in remote regions, and periodically reaches epidemic levels. Untreated HAT is always fatal, and the available drugs compromise toxicity and emerging resistance. The only safe treatment for late-stage disease is an inhibitor of an essential metabolic pathway that is involved in the synthesis of small organic cations termed polyamines. In this paper, we use genetic approaches to demonstrate how the parasite regulates this essential metabolic pathway. By modulating the protein levels of a trypanosome-specific activator of polyamine biosynthesis, the parasite has developed a mechanism to regulate pathway output. We also demonstrate that this pathway activator is essential to parasite growth. Our data strengthen the genetic and chemical validation of a key enzyme in this pathway as a drug target in the parasite, and they provide new insight into parasite-specific approaches that could be used to design novel drugs against this deadly disease.
PMCID: PMC2562514  PMID: 18949025
16.  Trypanosoma brucei adenine-phosphoribosyltransferases mediate adenine salvage and aminopurinol susceptibility but not adenine toxicity☆ 
Graphical abstract
•African trypanosomes possess two distinct adenine phosphoribosyltransferases.•Trypanosoma brucei TbAPRT1 is cytosolic, TbAPRT2 localizes to the glycosome.•Aprt1,2 null mutants are viable but do not incorporate adenine into nucleotides.•Aprt1,2 null mutants are resistant to aminopurinol but still sensitive to adenine.•Aminopurinol is a trypanocide with submicromolar activity against T. brucei.
African trypanosomes, like all obligate parasitic protozoa, cannot synthesize purines de novo and import purines from their hosts to build nucleic acids. The purine salvage pathways of Trypanosoma brucei being redundant, none of the involved enzymes is likely to be essential. Nevertheless they can be of pharmacological interest due to their role in activation of purine nucleobase or nucleoside analogues, which only become toxic when converted to nucleotides. Aminopurine antimetabolites, in particular, are potent trypanocides and even adenine itself is toxic to trypanosomes at elevated concentrations. Here we report on the T. brucei adenine phosphoribosyltransferases TbAPRT1 and TbAPRT2, encoded by the two genes Tb927.7.1780 and Tb927.7.1790, located in tandem on chromosome seven. The duplication is syntenic in all available Trypanosoma genomes but not in Leishmania. While TbAPRT1 is cytosolic, TbAPRT2 possesses a glycosomal targeting signal and co-localizes with the glycosomal marker aldolase. Interestingly, the distribution of glycosomal targeting signals among trypanosomatid adenine phosphoribosyltransferases is not consistent with their phylogeny, indicating that the acquisition of adenine salvage to the glycosome happened after the radiation of Trypanosoma. Double null mutant T. brucei Δtbaprt1,2 exhibited no growth phenotype but no longer incorporated exogenous adenine into the nucleotide pool. This, however, did not reduce their sensitivity to adenine. The Δtbaprt1,2 trypanosomes were resistant to the adenine isomer aminopurinol, indicating that it is activated by phosphoribosyl transfer. Aminopurinol was about 1000-fold more toxic to bloodstream-form T. brucei than the corresponding hypoxanthine isomer allopurinol. Aminopurinol uptake was not dependent on the aminopurine permease P2 that has been implicated in drug resistance.
PMCID: PMC3940079  PMID: 24596669
Adenine phosphoribosyltransferase; African trypanosomes; Purine salvage; Aminopurinol
17.  Regulation of Phosphatidylethanolamine Homeostasis—The Critical Role of CTP:Phosphoethanolamine Cytidylyltransferase (Pcyt2) 
Phosphatidylethanolamine (PE) is the most abundant lipid on the protoplasmatic leaflet of cellular membranes. It has a pivotal role in cellular processes such as membrane fusion, cell cycle regulation, autophagy, and apoptosis. CTP:phosphoethanolamine cytidylyltransferase (Pcyt2) is the main regulatory enzyme in de novo biosynthesis of PE from ethanolamine and diacylglycerol by the CDP-ethanolamine Kennedy pathway. The following is a summary of the current state of knowledge on Pcyt2 and how splicing and isoform specific differences could lead to variations in functional properties in this family of enzymes. Results from the most recent studies on Pcyt2 transcriptional regulation, promoter function, autophagy, and cell growth regulation are highlighted. Recent data obtained from Pcyt2 knockout mouse models is also presented, demonstrating the essentiality of this gene in embryonic development as well as the major physiological consequences of deletion of one Pcyt2 allele. Those include development of symptoms of the metabolic syndrome such as elevated lipogenesis and lipoprotein secretion, hypertriglyceridemia, liver steatosis, obesity, and insulin resistance. The objective of this review is to elucidate the nature of Pcyt2 regulation by linking its catalytic function with the regulation of lipid and energy homeostasis.
PMCID: PMC3588000  PMID: 23354482
phosphatidylethanolamine; CTP:phosphoethanolamine cytidylyltransferase; Pcyt2; lipid homeostasis; cell growth; hypertriglyceridemia; liver steatosis; obesity; insulin resistance; metabolic syndrome
18.  The Major Sites of Cellular Phospholipid Synthesis and Molecular Determinants of Fatty Acid and Lipid Head Group Specificity 
Molecular Biology of the Cell  2002;13(9):3148-3161.
Phosphatidylcholine and phosphatidylethanolamine are the two main phospholipids in eukaryotic cells comprising ∼50 and 25% of phospholipid mass, respectively. Phosphatidylcholine is synthesized almost exclusively through the CDP-choline pathway in essentially all mammalian cells. Phosphatidylethanolamine is synthesized through either the CDP-ethanolamine pathway or by the decarboxylation of phosphatidylserine, with the contribution of each pathway being cell type dependent. Two human genes, CEPT1 and CPT1, code for the total compliment of activities that directly synthesize phosphatidylcholine and phosphatidylethanolamine through the CDP-alcohol pathways. CEPT1 transfers a phosphobase from either CDP-choline or CDP-ethanolamine to diacylglycerol to synthesize both phosphatidylcholine and phosphatidylethanolamine, whereas CPT1 synthesizes phosphatidylcholine exclusively. We show through immunofluorescence that brefeldin A treatment relocalizes CPT1, but not CEPT1, implying CPT1 is found in the Golgi. A combination of coimmunofluorescence and subcellular fractionation experiments with various endoplasmic reticulum, Golgi, and nuclear markers confirmed that CPT1 was found in the Golgi and CEPT1 was found in both the endoplasmic reticulum and nuclear membranes. The rate-limiting step for phosphatidylcholine synthesis is catalyzed by the amphitropic CTP:phosphocholine cytidylyltransferase α, which is found in the nucleus in most cell types. CTP:phosphocholine cytidylyltransferase α is found immediately upstream cholinephosphotransferase, and it translocates from a soluble nuclear location to the nuclear membrane in response to activators of the CDP-choline pathway. Thus, substrate channeling of the CDP-choline produced by CTP:phosphocholine cytidylyltransferase α to nuclear located CEPT1 is the mechanism by which upregulation of the CDP-choline pathway increases de novo phosphatidylcholine biosynthesis. In addition, a series of CEPT1 site-directed mutants was generated that allowed for the assignment of specific amino acid residues as structural requirements that directly alter either phospholipid head group or fatty acyl composition. This pinpointed glycine 156 within the catalytic motif as being responsible for the dual CDP-alcohol specificity of CEPT1, whereas mutations within helix 214–228 allowed for the orientation of transmembrane helices surrounding the catalytic site to be definitively positioned.
PMCID: PMC124149  PMID: 12221122
19.  Lipidomic analysis of bloodstream and procyclic form Trypanosoma brucei 
Parasitology  2010;137(9):1357-1392.
The biological membranes of Trypanosoma brucei contain a complex array of phospholipids that are synthesized de novo from precursors obtained either directly from the host, or as catabolised endocytosed lipids. This paper describes the use of nanoflow electrospray tandem mass spectrometry and high resolution mass spectrometry in both positive and negative ion modes, allowing the identification of ~500 individual molecular phospholipids species from total lipid extracts of cultured bloodstream and procyclic form T. brucei. Various molecular species of all of the major subclasses of glycerophospholipids were identified including phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol as well as phosphatidic acid, phosphatidylglycerol and cardolipin, and the sphingolipids sphingomyelin, inositol phosphoceramide and ethanolamine phosphoceramide. The lipidomic data obtained in this study will aid future biochemical phenotyping of either genetically or chemically manipulated commonly used bloodstream and procyclic strains of Trypanosoma brucei. Hopefully this will allow a greater understanding of the bizarre world of lipids in this important human pathogen.
PMCID: PMC3744936  PMID: 20602846
Phospholipid; Trypanosoma brucei; mass spectrometry; lipidomics
20.  Meclizine Inhibits Mitochondrial Respiration through Direct Targeting of Cytosolic Phosphoethanolamine Metabolism* 
The Journal of Biological Chemistry  2013;288(49):35387-35395.
Background: Previous studies have shown that meclizine inhibits respiration in intact cells, but not in isolated mitochondria, via an unknown mechanism.
Results: Meclizine directly inhibits PCYT2 (CTP:phosphoethanolamine cytidylyltransferase).
Conclusion: Meclizine attenuates mitochondrial respiration by directly inhibiting the Kennedy pathway of phosphatidylethanolamine biosynthesis.
Significance: We identified a novel molecular target of meclizine, an over-the-counter antinausea drug, raising possibilities for new clinical applications.
We recently identified meclizine, an over-the-counter drug, as an inhibitor of mitochondrial respiration. Curiously, meclizine blunted respiration in intact cells but not in isolated mitochondria, suggesting an unorthodox mechanism. Using a metabolic profiling approach, we now show that treatment with meclizine leads to a sharp elevation of cellular phosphoethanolamine, an intermediate in the ethanolamine branch of the Kennedy pathway of phosphatidylethanolamine biosynthesis. Metabolic labeling and in vitro enzyme assays confirmed direct inhibition of the cytosolic enzyme CTP:phosphoethanolamine cytidylyltransferase (PCYT2). Inhibition of PCYT2 by meclizine led to rapid accumulation of its substrate, phosphoethanolamine, which is itself an inhibitor of mitochondrial respiration. Our work identifies the first pharmacologic inhibitor of the Kennedy pathway, demonstrates that its biosynthetic intermediate is an endogenous inhibitor of respiration, and provides key mechanistic insights that may facilitate repurposing meclizine for disorders of energy metabolism.
PMCID: PMC3853286  PMID: 24142790
Energy Metabolism; Metabolomics; Mitochondria; Phosphatidylethanolamine; Respiration; Meclizine; Phosphoethanolamine
21.  Crystallization and preliminary X-ray analysis of CTP:phosphoethanolamine cytidylyltransferase (ECT) from Saccharomyces cerevisiae  
CTP:phosphoethanolamine cytidylyltransferase from S. cerevisiae has been expressed, purified and crystallized.
CTP:phosphoethanolamine cytidylyltransferase (ECT) is the enzyme that catalyzes the conversion of phosphoethanolamine to CDP-ethanolamine in the phosphatidylethanolamine-biosynthetic pathway (Kennedy pathway). ECT from Saccharomyces cerevisiae was crystallized by the sitting-drop vapour-diffusion method using PEG 4000 as precipitant. The crystals diffracted X-­rays from a synchrotron-radiation source to 1.88 Å resolution. The space group was assigned as primitive tetragonal, P41212 or P43212, with unit-cell parameters a = b = 66.3, c = 150.8 Å. The crystals contain one ECT molecule in the asymmetric unit (V M = 2.2 Å3 Da−1), with a solvent content of 43%.
PMCID: PMC2225198  PMID: 17012796
CTP:phosphoethanolamine cytidylyltransferase; phosphatidylethanolamine-biosynthetic pathway; Saccharomyces cerevisiae
22.  Post-Transcriptional Regulation of the Trypanosome Heat Shock Response by a Zinc Finger Protein 
PLoS Pathogens  2013;9(4):e1003286.
In most organisms, the heat-shock response involves increased heat-shock gene transcription. In Kinetoplastid protists, however, virtually all control of gene expression is post-transcriptional. Correspondingly, Trypanosoma brucei heat-shock protein 70 (HSP70) synthesis after heat shock depends on regulation of HSP70 mRNA turnover. We here show that the T. brucei CCCH zinc finger protein ZC3H11 is a post-transcriptional regulator of trypanosome chaperone mRNAs. ZC3H11 is essential in bloodstream-form trypanosomes and for recovery of insect-form trypanosomes from heat shock. ZC3H11 binds to mRNAs encoding heat-shock protein homologues, with clear specificity for the subset of trypanosome chaperones that is required for protein refolding. In procyclic forms, ZC3H11 was required for stabilisation of target chaperone-encoding mRNAs after heat shock, and the HSP70 mRNA was also decreased upon ZC3H11 depletion in bloodstream forms. Many mRNAs bound to ZC3H11 have a consensus AUU repeat motif in the 3′-untranslated region. ZC3H11 bound preferentially to AUU repeats in vitro, and ZC3H11 regulation of HSP70 mRNA in bloodstream forms depended on its AUU repeat region. Tethering of ZC3H11 to a reporter mRNA increased reporter expression, showing that it is capable of actively stabilizing an mRNA. These results show that expression of trypanosome heat-shock genes is controlled by a specific RNA-protein interaction. They also show that heat-shock-induced chaperone expression in procyclic trypanosome enhances parasite survival at elevated temperatures.
Author Summary
When organisms are placed at a temperature that is higher than normal, their proteins start to unfold. The organisms protect themselves by increasing the synthesis of “heat-shock” proteins which can re-fold other proteins when the temperature returns to normal. In trypanosomes, the degradation of mRNAs that encode heat-shock proteins is slowed down at elevated temperatures. Trypanosoma brucei multiplies as “bloodstream forms” in the blood of mammals, at temperatures between 37–39°C; and as “procyclic forms” in Tsetse flies, which are usually at 20–37°C but can survive at 41°C. In this paper we show that in Trypanosoma brucei, a protein called ZC3H11 can bind to many heat-shock-protein mRNAs. ZC3H11 is essential in bloodstream-form trypanosomes and for recovery of procyclic-form trypanosomes after heat shock. ZC3H11 binds to an AUU repeat motif which is found in parts of the target mRNAs that do not encode protein. Several heat-shock-protein RNAs were decreased when we decreased the amount of ZC3H11 in bloodstream-form trypanosomes. These and other results show that expression of the specific subset of trypanosome heat-shock proteins is controlled by the interaction of ZC3H11 with the relevant mRNAs. They also show that the heat-shock response could enhance survival of trypanosomes in over-heated Tsetse flies.
PMCID: PMC3616968  PMID: 23592996
23.  Trypanin, a Component of the Flagellar Dynein Regulatory Complex, Is Essential in Bloodstream Form African Trypanosomes 
PLoS Pathogens  2006;2(9):e101.
The Trypanosoma brucei flagellum is a multifunctional organelle with critical roles in motility, cellular morphogenesis, and cell division. Although motility is thought to be important throughout the trypanosome lifecycle, most studies of flagellum structure and function have been restricted to the procyclic lifecycle stage, and our knowledge of the bloodstream form flagellum is limited. We have previously shown that trypanin functions as part of a flagellar dynein regulatory system that transmits regulatory signals from the central pair apparatus and radial spokes to axonemal dyneins. Here we investigate the requirement for this dynein regulatory system in bloodstream form trypanosomes. We demonstrate that trypanin is localized to the flagellum of bloodstream form trypanosomes, in a pattern identical to that seen in procyclic cells. Surprisingly, trypanin RNA interference is lethal in the bloodstream form. These knockdown mutants fail to initiate cytokinesis, but undergo multiple rounds of organelle replication, accumulating multiple flagella, nuclei, kinetoplasts, mitochondria, and flagellum attachment zone structures. These findings suggest that normal flagellar beat is essential in bloodstream form trypanosomes and underscore the emerging concept that there is a dichotomy between trypanosome lifecycle stages with respect to factors that contribute to cell division and cell morphogenesis. This is the first time that a defined dynein regulatory complex has been shown to be essential in any organism and implicates the dynein regulatory complex and other enzymatic regulators of flagellar motility as candidate drug targets for the treatment of African sleeping sickness.
African trypanosomes are protozoan parasites that cause African sleeping sickness, a fatal disease with devastating health and economic consequences. These parasites are indigenous to a 9 million-km2 area of sub-Saharan Africa where 60 million people live at risk of infection every day. In addition to the tremendous human health burden posed by trypanosomes, their infection of wild and domestic animals presents a barrier to sustained economic development of vast regions of otherwise productive land. Current drugs used for treatment of sleeping sickness are antiquated, toxic, and often ineffective; thus, there is a dire need for the development of innovative approaches for therapeutic intervention. Trypanosomes are highly motile and this motility requires coordinated regulation of axonemal dynein, a molecular motor that drives beating of the parasite's flagellum. In the present work, the authors demonstrate that the protein trypanin, which is part of a signaling system that regulates the flagellar dynein motor, is essential in bloodstream stage African trypanosomes. This surprising finding raises the possibility that numerous enzymes and regulatory proteins that are necessary for flagellar motility may represent novel targets for chemotherapeutic intervention in African sleeping sickness.
PMCID: PMC1579245  PMID: 17009870
24.  Clathrin-Dependent Targeting of Receptors to the Flagellar Pocket of Procyclic-Form Trypanosoma brucei 
Eukaryotic Cell  2004;3(4):1004-1014.
In trypanosomatids, endocytosis and exocytosis occur exclusively at the flagellar pocket, which represents about 0.43% of the pellicle membrane and is a deep invagination of the plasma membrane where the flagellum extends from the cell. Receptor molecules are selectively retained at the flagellar pocket. We studied the function of clathrin heavy chain (TbCLH) in the trafficking of the flagellar pocket receptors in Trypanosoma brucei by using the double-stranded RNA interference approach. It appears that TbCLH is essential for the survival of both the procyclic form and the bloodstream form of T. brucei, even though structures resembling large coated endocytic vesicles are absent in procyclic-form trypanosomes. Down-regulation of TbCLH by RNA interference (RNAi) for 24 h rapidly and drastically reduced the uptake of macromolecules via receptor-mediated endocytosis in procyclic-form trypanosomes. This result suggested the importance of TbCLH in receptor-mediated endocytosis of the procyclic-form trypanosome, in which the formation of large coated endocytic vesicles may not be required. Surprisingly, induction of TbCLH RNAi in the procyclic T. brucei for a period of 48 h prohibited the export of the flagellar pocket-associated transmembrane receptor CRAM from the endoplasmic reticulum to the flagellar pocket, while trafficking of the glycosylphosphatidylinositol-anchored procyclin coat was not significantly affected. After 72 h of induction of TbCLH RNAi, procyclics exhibited morphological changes to an apolar round shape without a distinct structure of the flagellar pocket and flagellum. Although trypanosomes, like other eukaryotes, use similar organelles and machinery for protein sorting and transport, our studies reveal a novel role for clathrin in the secretory pathway of trypanosomes. We speculate that the clathrin-dependent trafficking of proteins to the flagellar pocket may be essential for the biogenesis and maintenance of the flagellar pocket in trypanosomes.
PMCID: PMC500874  PMID: 15302833
25.  A Function for a Specific Zinc Metalloprotease of African Trypanosomes 
PLoS Pathogens  2007;3(10):e150.
The Trypanosoma brucei genome encodes three groups of zinc metalloproteases, each of which contains ∼30% amino acid identity with the major surface protease (MSP, also called GP63) of Leishmania. One of these proteases, TbMSP-B, is encoded by four nearly identical, tandem genes transcribed in both bloodstream and procyclic trypanosomes. Earlier work showed that RNA interference against TbMSP-B prevents release of a recombinant variant surface glycoprotein (VSG) from procyclic trypanosomes. Here, we used gene deletions to show that TbMSP-B and a phospholipase C (GPI-PLC) act in concert to remove native VSG during differentiation of bloodstream trypanosomes to procyclic form. When the four tandem TbMSP-B genes were deleted from both chromosomal alleles, bloodstream B −/− trypanosomes could still differentiate to procyclic form, but VSG was removed more slowly and in a non-truncated form compared to differentiation of wild-type organisms. Similarly, when both alleles of the single-copy GPI-PLC gene were deleted, bloodstream PLC −/− cells could still differentiate. However, when all the genes for both TbMSP-B and GPI-PLC were deleted from the diploid genome, the bloodstream B −/− PLC −/− trypanosomes did not proliferate in the differentiation medium, and 60% of the VSG remained on the cell surface. Inhibitors of cysteine proteases did not affect this result. These findings demonstrate that removal of 60% of the VSG during differentiation from bloodstream to procyclic form is due to the synergistic activities of GPI-PLC and TbMSP-B.
Author Summary
African trypanosomes cause sleeping sickness, a fatal disease of humans and livestock in Africa. During their life cycle, these protozoan parasites cycle between the bloodstream of mammals and tsetse flies. Their two main developmental stages are the bloodstream form and the procyclic form in the tsetse fly. Bloodstream trypanosomes thwart their host's immune response by periodically switching their major surface protein, the variant surface glycoprotein (VSG). When bloodstream trypanosomes are ingested by a tsetse fly, they must quickly shed the VSG and replace it with an unrelated invariant protein more suited to their survival as procyclic organisms in the fly midgut. Here, we examine the mechanisms used by trypanosomes to remove the VSG during their differentiation from bloodstream to procyclic form in culture. We deleted the genes for one of the trypanosome's protease enzymes from the trypanosome genome and found that bloodstream trypanosomes could still differentiate to the procyclic form, but VSG removal was diminished. Deleting the genes for a phospholipase enzyme had a similar effect—they could still differentiate but VSG removal was impaired. When the genes for both the protease and the phospholipase were deleted, bloodstream trypanosomes could not differentiate to the procyclic form, they retained about 60% of the VSG on their surface, and they died in the differentiation medium. These results highlight the synergistic roles of these two enzymes in the differentiation process.
PMCID: PMC2034397  PMID: 17953481

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