Trypanosoma brucei brucei: in vitro production of metacyclic forms.

An in vitro method has been established to obtain metacyclic form populations of Trypanosoma brucei brucei. Trypanosome populations containing more than 98% of metacyclic forms were obtained from cultures which were: 1) initiated with bloodstream forms in primary cultures in the presence of Microtus montanus embryonic fibroblast-like cells (feeder cell layers); 2) maintained in glucose-free Eagle's minimum essential medium supplemented with 10 mM L-proline, 2 mM L-glutamine and 20% (v/v) fetal bovine serum at 27 degrees C without medium change for five days; 3) subcultured in the absence of the feeder cell layers but in the presence of Cytodex 3 beads; 4) maintained for an additional nine days with medium changes on days 5, 8 and 11; and 5) harvested on day 14 by means of diethylaminoethyl cellulose column chromatography prior to the appearance of other infective forms. Most of the trypanosomes obtained under these conditions were morphologically similar to metacyclic forms derived from tsetse fly vectors, coated with variable surface glycoprotein and were infective for mice. In the primary cultures procyclic forms, epimastigotes and metacyclic forms appeared by day 8. When the duration of the subculture was prolonged to 17 days or more at 27 degrees C, the metacyclic forms decreased in number while short trypomastigotes, long slender epimastigotes, and long slender trypomastigotes increased in number. These forms in such long-term cultures also appeared in diethylaminoethyl cellulose-isolated populations along with metacyclic forms.     

Nucleoside diphosphate kinase of Trypanosoma brucei

Nucleoside diphosphate kinase (NDPK) is a highly conserved, multifunctional enzyme. Its originally described function is the phosphorylation of nucleoside diphosphates to the corresponding triphosphates, using ATP as the phosphate donor and a high-energy phosphorylated histidine residue as the reaction intermediate. More recently, a host of additional functions of NDPK have been discovered. Some of these correlate with the capacity of NDPK to transphosphorylate other proteins, in a manner reminiscent of bacterial two-component systems. Other functions may be mediated by direct DNA-binding of NDPK.This study describes the identification of NDPK from the parasitic protozoon Trypanosoma brucei. The genome of this major disease agent contains a single gene for NDPK. The predicted amino acid sequence of the trypanosomal enzyme is highly conserved with respect to all other species. The protein is constitutively expressed and is present in procyclic and in bloodstream forms. Immunofluorescence and immuno-electron microscopy demonstrate that trypanosomal NDPK (TbNDPK) is predominantly localized in the cell nucleus. Histidine phosphorylation of TbNDPK is essentially resistant to the experimental compound LY266500, a potent inhibitor of histidine phosphorylation of trypanosomal succinyl coenzyme A synthase.     

The ASCT/SCS cycle fuels mitochondrial ATP and acetate production in Trypanosoma brucei

Acetate:succinate CoA transferase (ASCT) is a mitochondrial enzyme that catalyzes the production of acetate and succinyl-CoA, which is coupled to ATP production with succinyl-CoA synthetase (SCS) in a process called the ASCT/SCS cycle. This cycle has been studied in Trypanosoma brucei (T. brucei), a pathogen of African sleeping sickness, and is involved in (i) ATP and (ii) acetate production and proceeds independent of oxygen and an electrochemical gradient. Interestingly, knockout of ASCT in procyclic form (PCF) of T. brucei cause oligomycin A-hypersensitivity phenotype indicating that ASCT/SCS cycle complements the deficiency of ATP synthase activity. In bloodstream form (BSF) of T. brucei, ATP synthase works in reverse to maintain the electrochemical gradient by hydrolyzing ATP. However, no information has been available on the source of ATP, although ASCT/SCS cycle could be a potential candidate. Regarding mitochondrial acetate production, which is essential for fatty acid biosynthesis and growth of T. brucei, ASCT or acetyl-CoA hydrolase (ACH) are known to be its source. Despite the importance of this cycle, direct evidence of its function is lacking, and there are no comprehensive biochemical or structural biology studies reported so far. Here, we show that in vitro–reconstituted ASCT/SCS cycle is highly specific towards acetyl-CoA and has a higher k_cat than that of yeast and bacterial ATP synthases. Our results provide the first biochemical basis for (i) rescue of ATP synthase-deficient phenotype by ASCT/SCS cycle in PCF and (ii) a potential source of ATP for the reverse reaction of ATP synthase in BSF.     

Contribution of Pyruvate Phosphate Dikinase in the Maintenance of the Glycosomal ATP/ADP Balance in the Trypanosoma brucei Procyclic Form

Trypanosoma brucei belongs to a group of protists that sequester the first six or seven glycolytic steps inside specialized peroxisomes, named glycosomes. Because of the glycosomal membrane impermeability to nucleotides, ATP molecules consumed by the first glycolytic steps need to be regenerated in the glycosomes by kinases, such as phosphoenolpyruvate carboxykinase (PEPCK). The glycosomal pyruvate phosphate dikinase (PPDK), which reversibly converts phosphoenolpyruvate into pyruvate, could also be involved in this process. To address this question, we analyzed the metabolism of the main carbon sources used by the procyclic trypanosomes (glucose, proline, and threonine) after deletion of the PPDK gene in the wild-type (Δppdk) and PEPCK null (Δppdk/Δpepck) backgrounds. The rate of acetate production from glucose is 30% reduced in the Δppdk mutant, whereas threonine-derived acetate production is not affected, showing that PPDK function in the glycolytic direction with production of ATP in the glycosomes. The Δppdk/Δpepck mutant incubated in glucose as the only carbon source showed a 3.8-fold reduction of the glycolytic rate compared with the Δpepck mutant, as a consequence of the imbalanced glycosomal ATP/ADP ratio. The role of PPDK in maintenance of the ATP/ADP balance was confirmed by expressing the glycosomal phosphoglycerate kinase (PGKC) in the Δppdk/Δpepck cell line, which restored the glycolytic flux. We also observed that expression of PGKC is lethal for procyclic trypanosomes, as a consequence of ATP depletion, due to glycosomal relocation of cytosolic ATP production. This illustrates the key roles played by glycosomal and cytosolic kinases, including PPDK, to maintain the cellular ATP/ADP homeostasis.     

A deletion site editing endonuclease in Trypanosoma brucei

RNA editing in Trypanosoma brucei inserts and deletes uridines in mitochondrial mRNAs by a series of enzymatic steps that are catalyzed by a multiprotein complex, the editosome. KREPB1 and two related editosome proteins KREPB2 and KREPB3 contain motifs that suggest endonuclease and RNA/protein interaction functions. Repression of KREPB1 expression in procyclic forms by RNAi inhibited growth, in vivo editing, and in vitro endoribonucleolytic cleavage of deletion substrates. However, cleavage of insertion substrates and the exoUase, TUTase, and ligase catalytic activities of editing were retained by 20S editosomes. Repression of expression of an ectopic KREPB1 allele in bloodstream forms lacking both endogenous alleles or exclusive expression of KREPB1 with point mutations in the putative RNase III catalytic domain also blocked growth, in vivo editing, and abolished cleavage of deletion substrates, without affecting the other editing steps. These data indicate that KREPB1 is an endoribonuclease that is specific for RNA editing deletion sites.     

Ethyl Pyruvate Emerges as a Safe and Fast Acting Agent against Trypanosoma brucei by Targeting Pyruvate Kinase Activity

Background

Human African Trypanosomiasis (HAT) also called sleeping sickness is an infectious disease in humans caused by an extracellular protozoan parasite. The disease, if left untreated, results in 100% mortality. Currently available drugs are full of severe drawbacks and fail to escape the fast development of trypanosoma resistance. Due to similarities in cell metabolism between cancerous tumors and trypanosoma cells, some of the current registered drugs against HAT have also been tested in cancer chemotherapy. Here we demonstrate for the first time that the simple ester, ethyl pyruvate, comprises such properties.

Results

The current study covers the efficacy and corresponding target evaluation of ethyl pyruvate on T. brucei cell lines using a combination of biochemical techniques including cell proliferation assays, enzyme kinetics, phasecontrast microscopic video imaging and ex vivo toxicity tests. We have shown that ethyl pyruvate effectively kills trypanosomes most probably by net ATP depletion through inhibition of pyruvate kinase (Ki = 3.0±0.29 mM). The potential of ethyl pyruvate as a trypanocidal compound is also strengthened by its fast acting property, killing cells within three hours post exposure. This has been demonstrated using video imaging of live cells as well as concentration and time dependency experiments. Most importantly, ethyl pyruvate produces minimal side effects in human red cells and is known to easily cross the blood-brain-barrier. This makes it a promising candidate for effective treatment of the two clinical stages of sleeping sickness. Trypanosome drug-resistance tests indicate irreversible cell death and a low incidence of resistance development under experimental conditions.

Conclusion

Our results present ethyl pyruvate as a safe and fast acting trypanocidal compound and show that it inhibits the enzyme pyruvate kinase. Competitive inhibition of this enzyme was found to cause ATP depletion and cell death. Due to its ability to easily cross the blood-brain-barrier, ethyl pyruvate could be considered as new candidate agent to treat the hemolymphatic as well as neurological stages of sleeping sickness.     

Stage-specific requirement of a mitogen-activated protein kinase by Trypanosoma brucei

In cycling between the mammalian host and the tsetse fly vector, African trypanosomes undergo adaptive differentiation steps that are coupled to growth control. The signaling pathways underlying these cellular processes are largely unknown. Mitogen-activated protein kinases (MAPKs) are known mediators of growth and differentiation in other eukaryotic organisms. To establish the function of a MAPK homologue, TbMAPK2, in T. brucei, a null mutant was constructed. Bloodstream forms of a deltamapk2/deltamapk2 clone were able to grow normally and exhibited no detectable phenotype. When these cells were triggered to differentiate in vitro, however, they developed to the procyclic (fly midgut) form with delayed kinetics and subsequently underwent cell cycle arrest. Introduction of an ectopic copy of the TbMAPK2 gene into the null mutant restored its ability to differentiate and to divide. In contrast, a TbMAPK2 mutant, in which the T190 and Y192 residues of the activating phosphorylation site were replaced by A and F, was unable to restore the growth and differentiation phenotypes. Analysis of the DNA content and the nucleus/kinetoplast configuration of individual cells showed that the null mutant was arrested in all phases of the cell cycle and that 25-30% of the cells had failed to segregate their nucleus and kinetoplast correctly. This implies that cell cycle progression by the procyclic form depends on a constitutive stimulus exerted by the signaling cascade operating through TbMAPK2.     

Differential effects of interferon-gamma on production of trypanosome-derived lymphocyte-triggering factor by Trypanosoma brucei gambiense and Trypanosoma brucei brucei

Trypanosome-derived lymphocyte-triggering factor (TLTF) produced by Trypanosoma brucei brucei stimulates production of interferon-gamma (IFN-gamma) by CD8+ T cells, and it is reported that, in turn, IFN-gamma stimulates proliferation of T. b. brucei. We studied the role of TLTF in trypanosome proliferation using the Wellcome strain (WS) of Trypanosoma brucei gambiense and the ILtat 1.4 strain (IL) of T. b. brucei. Increase in the number of WS in infected rats is more rapid than IL and corresponds with comparatively higher levels of IFN-gamma. Production of IFN-gamma, as measured by protein and messenger RNA (mRNA) levels, was maintained by splenocytes from WS-infected rats, whereas levels decreased in IL-infected rats, accompanied by prolongation of infection. Expression of TLTF mRNA by in vitro-cultured WS was promoted in a dose-dependent fashion by addition of recombinant rat IFN-gamma at all concentrations tested. The addition of lower concentrations of IFN-gamma to cultured IL increased expression of TLTF mRNA, whereas, in contrast to WS, addition of 100 and 1,000 U/ml IFN-gamma decreased expression of TLTF by IL. These results show that unlike WS, elevated IFN-gamma concentrations lead to decreased TLTF production by IL. It is believed that decreased TLTF production in IL-infected rats leads to lowered IFN-gamma production, thereby slowing IL proliferation.     

Characterization of a highly diverged mitochondrial ATP synthase Fo subunit in Trypanosoma brucei

The mitochondrial F₁F_o ATP synthase of the parasite Trypanosoma brucei has been previously studied in detail. This unusual enzyme switches direction in functionality during the life cycle of the parasite, acting as an ATP synthase in the insect stages, and as an ATPase to generate mitochondrial membrane potential in the mammalian bloodstream stages. Whereas the trypanosome F₁ moiety is relatively highly conserved in structure and composition, the F_o subcomplex and the peripheral stalk have been shown to be more variable. Interestingly, a core subunit of the latter, the normally conserved subunit b, has been resistant to identification by sequence alignment or biochemical methods. Here, we identified a 17 kDa mitochondrial protein of the inner membrane, Tb927.8.3070, that is essential for normal growth, efficient oxidative phosphorylation, and membrane potential maintenance. Pull-down experiments and native PAGE analysis indicated that the protein is both associated with the F₁F_o ATP synthase and integral to its assembly. In addition, its knockdown reduced the levels of F_o subunits, but not those of F₁, and disturbed the cell cycle. Finally, analysis of structural homology using the HHpred algorithm showed that this protein has structural similarities to F_o subunit b of other species, indicating that this subunit may be a highly diverged form of the elusive subunit b.     

The role of compartmentation and glycerol kinase in the synthesis of ATP within the glycosome of Trypanosoma brucei

Glycosomes, purified from trypomastigote forms of Trypanosoma brucei, contained all the enzymes necessary to convert glucose to alpha-glycerophosphate and 3-phosphoglycerate. The multienzyme reaction which produces 2 alpha-glycerophosphate, 2 ADP, and 2 NAD+ from 1 glucose, 2 ATP, and 2 NADH was studied spectrophotometrically. Intact glycosomes, suspended with 5.6 mM alpha-glycerophosphate and 2 mM ADP, produced ATP inside the glycosomes for glucose phosphorylation at a rate of 0.7 mumol/min/mg protein, so confirming the feasibility of producing ATP from alpha-glycerophosphate and ADP catalyzed by glycosomal glycerol kinase, and coupling this ATP production to the ATP-requiring stages of glycolysis. No evidence was found for direct channeling of the ATP generated by glycerol kinase and either hexokinase or phosphofructose kinase in glycosomal enzyme complexes cross-linked by dimethyl suberimidate treatment of intact glycosomes prior to solubilization of their membrane. Compartmentation of glycolytic intermediates, enzymes, and ATP inside isolated glycosomes was demonstrated by their inaccessibility to exogenous enzymes. We conclude that the compartmentation of the glycosome and the efficient production of ATP in the glycosome from whole cell concentrations of sn-glycerol 3-phosphate and ADP account for the observed whole cell production of equimolar glycerol from glucose with net ATP synthesis by T. brucei under anaerobic conditions.     

Purification and characterization of pyruvate kinase from Trypanosoma brucei

Pyruvate kinase (ATP: pyruvate phosphotransferase, EC 2.7.1.40) from Trypanosoma brucei has been partially purified by carboxymethylcellulose chromatography, and gel filtration. The enzyme is unstable in aqueous solution and requires the presence of a thiol protecting reagent as well as glycerol for the maintenance of activity. Dithiothreitol activates as well as stabilizes the enzyme. Phosphoenolpyruvate allosterically activates trypanosome pyruvate kinase whereas hyperbolic kinetics are found when ADP is the variable substrate. Mg²⁺ or Mn²⁺ ions and a monovalent cation are essential for enzyme activity. Fructose 1,6-diphosphate acts as a heterotropic allosteric activator, markedly decreasing the S_0.5 value for phosphoenolpyruvate from 1.34 to 0.25 mm at 1 mm fructose 1,6-diphosphate and transforms the phosphoenolpyruvate saturation curve from a sigmoidal to a hyperbolic form. The enzyme has a pH optimum of 6.5–7.0 and a molecular weight of 270,000 ± 27,000 as estimated by gel chromatography. Purine nucleotides are the preferred coenzymes for the reaction, having much lower K_m values than the pyrimidine nucleotides. The possible role of pyruvate kinase in the regulation of glycolysis in T. brucei is discussed.     

Adenosine kinase mediates high affinity adenosine salvage in Trypanosoma brucei

African sleeping sickness is caused by Trypanosoma brucei. This extracellular parasite lacks de novo purine biosynthesis, and it is therefore dependent on exogenous purines such as adenosine that is taken up from the blood and other body fluids by high affinity transporters. The general belief is that adenosine needs to be cleaved to adenine inside the parasites in order to be used for purine nucleotide synthesis. We have found that T. brucei also can salvage this nucleoside by adenosine kinase (AK), which has a higher affinity to adenosine than the cleavage-dependent pathway. The recombinant T. brucei AK (TbAK) preferably used ATP or GTP to phosphorylate both natural and synthetic nucleosides in the following order of catalytic efficiencies: adenosine > cordycepin > deoxyadenosine > adenine arabinoside (Ara-A) > inosine > fludarabine (F-Ara-A). TbAK differed from the AK of the related intracellular parasite Leishmania donovani by having a high affinity to adenosine (K m = 0.04-0.08 microm depending on [phosphate]) and by being negatively regulated by adenosine (K i = 8-14 microm). These properties make the enzyme functionally related to the mammalian AKs, although a phylogenetic analysis grouped it together with the L. donovani enzyme. The combination of a high affinity AK and efficient adenosine transporters yields a strong salvage system in T. brucei, a potential Achilles' heel making the parasites more sensitive than mammalian cells to adenosine analogs such as Ara-A. Studies of wild-type and AK knockdown trypanosomes showed that Ara-A inhibited parasite proliferation and survival in an AK-dependent manner by affecting nucleotide levels and by inhibiting nucleic acid biosynthesis.     

Microbody phosphoglycerate kinase of Trypanosoma brucei: expression and complementation in Escherichia coli

In the primitive eukaryotic parasite, Trypanosoma brucei, most of the enzymes of glycolysis are located within microbody organelles called glycosomes. Proteins destined for the glycosome are synthesized on free ribosomes and post-translationally translocated into the organelle. The gene, gPGK, encoding the glycosomal isozyme of phosphoglycerate kinase (gPGK), was cloned adjacent to a T7 promoter and cotransformed with a plasmid encoding T7 RNA polymerase into Escherichia coli Pgk-cells. Functional complementation occurred, but only after the creation of a ribosome-binding site by mutagenesis. This represents the first example of complementation of an E. coli mutant with a gene encoding a microbody protein. Enzymatically active recombinant gPGK was purified to near homogeneity by ion exchange chromatography from highly expressing E. coli. The recombinant protein will aid in studies of glycosomal biogenesis.     

Phospholipase A2 from Trypanosoma brucei gambiense and Trypanosoma brucei brucei: Inhibition by Organotins

Activity and kinetics of phospholipase A₂ (PLA₂) from Trypanosoma brucei gambiense (Wellcome strain) and Trypanosoma brucei brucei (GUTat 3.1) were examined using two different fluorescent substrates. The activity in the supernatants of sonicated parasites was Ca²⁺-independent, strongly stimulated by Triton X-100 with optimum activity at 37°C and pH 6.5–8.5. To encourage a possible interaction between the parasite enzyme and organotin compounds, fatty acid derivatives of dibutyltin dichloride were synthesized and evaluated as potential inhibitors of PLA₂. The enzyme from the two-trypanosome species differ with respect to kinetic parameters and are noncompetitively inhibited by the organotin compounds. The Michaelis constant (K_M) for PLA₂ from T. b. brucei is 63.87 and 30.90 μM while for T. b. gambiense it is 119.64 and 32.90 μM for the substrates l,2-bis-(1-pyrenebutanoyl-sn-glycero-3-phosphocholine (PBGPC) and 2-(12-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)dode-canoyl-1-hexadecanoyl-sn-glycero-3-phosphocholine (NBDC₁₂-HPC), respectively.     

Phospholipase A2 from Trypanosoma brucei gambiense and Trypanosoma brucei brucei: inhibition by organotins

Activity and kinetics of phospholipase A2 (PLA2) from Trypanosoma brucei gambiense (Wellcome strain) and Trypanosoma brucei brucei (GUTat 3.1) were examined using two different fluorescent substrates. The activity in the supernatants of sonicated parasites was Ca2+-independent, strongly stimulated by Triton X-100 with optimum activity at 37 degrees C and pH 6.5-8.5. To encourage a possible interaction between the parasite enzyme and organotin compounds, fatty acid derivatives of dibutyltin dichloride were synthesized and evaluated as potential inhibitors of PLA2. The enzyme from the two-trypanosome species differ with respect to kinetic parameters and are noncompetitively inhibited by the organotin compounds. The Michaelis constant (KM) for PLA2 from T. b. brucei is 63.87 and 30.90 microM while for T. b. gambiense it is 119.64 and 32.91 microM for the substrates 1,2-bis-(1-pyrenebutanoyl)-sn-glycero-3-phosphocholine (PBGPC) and 2-(12-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)dodecanoyl-1-hexadecanoyl-sn-glycero-3-phosphocholine (NBDC12-HPC), respectively.     

Biosynthesis of trypanothione in Trypanosoma brucei brucei

Trypanothione [T(SH)2], the major redox mediator in pathogenic trypanosomatids, is synthetized stepwise by two distinct enzymes in Crithidia fasciculata, while in Trypanosoma cruzi a single enzyme catalyzes both steps. A full-length reading frame presumed to encode trypanothione synthetase (TryS) was obtained by PCR using DNA of T. brucei as template and primers based on fragments of putative TryS genes. The recombinant protein produced by E. coli Origami (DE3) was purified to homogeneity by chelate and ion exchange chromatography. The enzyme catalyzed both reactions of T(SH)2 biosynthesis. Thus, T(SH)2 synthesis appears to be similar in African (T. brucei) and New World (T. cruzi) trypanosomes but distinct from that of Crithidia.     

Stimulation of Trypanosoma brucei pyruvate kinase by fructose 2,6-bisphosphate

The activity of pyruvate kinase present in a crude extract of the bloodstream form of Trypanosoma brucei was greatly increased by fructose 2,6-bisphosphate, which converted the saturation curve for phosphoenolpyruvate from a sigmoid into a hyperbola with no change in V. Phosphate and arsenate had an effect opposite to that of fructose 2,6-bisphosphate and the apparent Ka for fructose 2,6-bisphosphate was shifted from 75 nM to 1.5 microM by the presence of 5 mM phosphate. Fructose 1,6-bisphosphate had effects similar to those of fructose 2,6-bisphosphate but at approximately 4000-fold higher concentrations. Pyruvate kinases of Crithidia luciliae and of Leishmania major, two trypanosomatids which are like T. brucei in containing glycosomes, were also stimulated by fructose 2,6-bisphosphate and inhibited by phosphate.