Glucose-induced remodeling of intermediary and energy metabolism in procyclic Trypanosoma brucei

The procyclic form of Trypanosoma brucei is a parasitic protozoan that normally dwells in the midgut of its insect vector. In vitro, this parasite prefers d-glucose to l -proline as a carbon source, although this amino acid is the main carbon source available in its natural habitat. Here, we investigated how l -proline is metabolized in glucose-rich and glucose-depleted conditions. Analysis of the excreted end products of (13)C-enriched l -proline metabolism showed that the amino acid is converted into succinate or l -alanine depending on the presence or absence of d-glucose, respectively. The fact that the pathway of l -proline metabolism was truncated in glucose-rich conditions was confirmed by the analysis of 13 separate RNA interference-harboring or knock-out cell lines affecting different steps of this pathway. For instance, RNA interference studies revealed the loss of succinate dehydrogenase activity to be conditionally lethal only in the absence of d-glucose, confirming that in glucose-depleted conditions, l -proline needs to be converted beyond succinate. In addition, depletion of the F(0)/F(1)-ATP synthase activity by RNA interference led to cell death in glucose-depleted medium, but not in glucose-rich medium. This implies that, in the presence of d-glucose, the importance of the F(0)/F(1)-ATP synthase is diminished and ATP is produced by substrate level phosphorylation. We conclude that trypanosomes develop an elaborate adaptation of their energy production pathways in response to carbon source availability.     

Kinetics of methionine transport and metabolism by Trypanosoma brucei brucei and Trypanosoma brucei rhodesiense

Methionine is an essential amino acid for both prokaryotic and eukaryotic organisms; however, little is known concerning its utilization in African trypanosomes, protozoa of the Trypanosoma brucei group. This study explored the Michaelis-Menten kinetic constants for transport and pool formation as well as metabolic utilization of methionine by two divergent strains of African trypanosomes, Trypanosoma brucei brucei (a veterinary pathogen), highly sensitive to trypanocidal agents, and Trypanosoma brucei rhodesiense (a human pathogenic isolate), highly refractory to trypanocidal arsenicals. The Michaelis-Menten constants derived by Hanes-Woolf analysis for transport of methionine for T. b. brucei and T. b. rhodesiense, respectively, were as follows: K(M) values, 1. 15 and 1.75 mM; V(max) values, 3.97 x 10(-5) and 4.86 x 10(-5) mol/L/min. Very similar values were obtained by Lineweaver-Burk analysis (K(M), 0.25 and 1.0 mM; V(max), 1 x 10(-5) and 2.0 x 10(-5) mol/L/min, T. b. brucei and T. b. rhodesiense, respectively). Cooperativity analyses by Hill (log-log) plot gave Hill coefficients (n) of 6 and 2 for T. b. brucei and T. b. rhodesiense, respectively. Cytosolic accumulation of methionine after 10-min incubation with 25 mM exogenous methionine was 1.8-fold greater in T. b. rhodesiense than T. b. brucei (2.1 vs 1.1 mM, respectively). In African trypanosomes as in their mammalian host, S-adenosylmethionine (AdoMet) is the major product of methionine metabolism. Accumulation of AdoMet was measured by HPLC analysis of cytosolic extracts incubated in the presence of increasing cytosolic methionine. In trypanosomes incubated for 10 min with saturating methionine, both organisms accumulated similar amounts of AdoMet (approximately 23 microM), but the level of trans-sulfuration products (cystathionine and cysteine) in T. b. rhodesiense was double that of T. b. brucei. Methionine incorporation during protein synthesis in T. b. brucei was 2.5 times that of T. b. rhodesiense. These results further confirm our belief that the major pathways of methionine utilization, for polyamine synthesis, protein transmethylation and the trans-sulfuration pathway, are excellent targets for chemotherapeutic intervention against African trypanosomes.     

Adaptations in the lipid metabolism of the protozoan parasite Trypanosoma brucei

Trypanosomes are unicellular parasites and like all decent parasites, they try to obtain from the host as much material as possible, including lipids. However, the needs of a parasite are not always the same as those of the host, and therefore, mostly, some biosynthetic work still has to be done by the parasite itself. Very often at least modifications of the lipid components that are acquired from the host have to be made. Furthermore, next to the lipids Trypanosoma brucei indeed obtains from the host, some other lipid components have to be synthesized de novo. Especially the processes where the metabolism of T. brucei differs from that of the host, will be discussed, as at least some of them are excellent targets for the development of urgently needed new chemotherapeutics.     

Dynamic localization of Trypanosoma brucei mitochondrial DNA polymerase ID

Trypanosomes contain a unique form of mitochondrial DNA called kinetoplast DNA (kDNA) that is a catenated network composed of minicircles and maxicircles. Several proteins are essential for network replication, and most of these localize to the antipodal sites or the kinetoflagellar zone. Essential components for kDNA synthesis include three mitochondrial DNA polymerases TbPOLIB, TbPOLIC, and TbPOLID). In contrast to other kDNA replication proteins, TbPOLID was previously reported to localize throughout the mitochondrial matrix. This spatial distribution suggests that TbPOLID requires redistribution to engage in kDNA replication. Here, we characterize the subcellular distribution of TbPOLID with respect to the Trypanosoma brucei cell cycle using immunofluorescence microscopy. Our analyses demonstrate that in addition to the previously reported matrix localization, TbPOLID was detected as discrete foci near the kDNA. TbPOLID foci colocalized with replicating minicircles at antipodal sites in a specific subset of the cells during stages II and III of kDNA replication. Additionally, the TbPOLID foci were stable following the inhibition of protein synthesis, detergent extraction, and DNase treatment. Taken together, these data demonstrate that TbPOLID has a dynamic localization that allows it to be spatially and temporally available to perform its role in kDNA replication.     

Measure of molecular diversity within the Trypanosoma brucei subspecies Trypanosoma brucei brucei and Trypanosoma brucei gambiense as revealed by genotypic characterization

We have evaluated whether sequence polymorphisms in the rRNA intergenic spacer region can be used to study the relatedness of two subspecies of Trypanosoma brucei. Thirteen T. brucei isolates made up of 6 T. b. brucei and 7 T. b. gambiense were analyzed using restriction fragment length polymorphism (RFLP). By PCR-based restriction mapping of the ITS1-5.8S-ITS2 ribosomal repeat unit, we found a fingerprint pattern that separately identifies each of the two subspecies analyzed, with unique restriction fragments observed in all but 1 of the T. b. gambiense "human" isolates. Interestingly, the restriction profile for a virulent group 2 T. b. gambiense human isolate revealed an unusual RFLP pattern different from the profile of other human isolates. Sequencing data from four representatives of each of the two subspecies indicated that the intergenic spacer region had a conserved ITS-1 and a variable 5.8S with unique transversions, insertions, or deletions. The ITS-2 regions contained a single repeated element at similar positions in all isolates examined, but not in 2 of the human isolates. A unique 4-bp [C(3)A] sequence was found within the 5.8S region of human T. b. gambiense isolates. Phylogenetic analysis of the data suggests that their common ancestor was a nonhuman animal pathogen and that human pathogenicity might have evolved secondarily. Our data show that cryptic species within the T. brucei group can be distinguished by differences in the PCR-RFLP profile of the rDNA repeat.     

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.     

Energy generation in insect stages of Trypanosoma brucei: metabolism in flux

The generation of energy in African trypanosomes is a subject of undoubted importance. In bloodstream-form organisms, substrate-level phosphorylation of glucose is sufficient to provide the energy needs of the parasite. The situation in procyclic-form trypanosomes is more complex. For many years, it was accepted that glucose metabolism followed a conventional scheme involving glycolysis, the tricarboxylic acid cycle and ATP-producing oxidative phosphorylation linked to the electron-transport chain. However, progress in sequencing the Trypanosoma brucei genome and the development of gene-knockout and RNA interference technology has provided novel insight. Coupling these new technologies with classical approaches, including NMR and mass spectrometry to analyse glycolytic intermediates and end products, has yielded several surprises. In this article, we summarize how these recent data have helped to change the view of metabolism in procyclic-form T. brucei.     

Differential effects of Trypanosoma brucei gambiense and Trypanosoma brucei brucei on rat macrophages

Mammalian immune responses to Trypanosoma brucei infection are important to control of the disease. In rats infected with T. brucei gambiense (Wellcome strain; WS) or T. brucei brucei (interleukin-tat 1.4 strain [ILS]), a marked increase in the number of macrophages in the spleen can be observed. However, the functional repercussions related to this expansion are not known. To help uncover the functional significance of macrophages in the context of trypanosome infection, we determined the mRNA levels of genes associated with an increase in macrophage number or macrophage function in WS- and ILS-infected rats and in cultured cells. Specifically, we assayed mRNA levels for macrophage colony stimulating factor (M-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), and macrophage migration inhibitory factor (MIF). Upregulation of GM-CSF and MIF mRNA levels was robust in comparison with changes in M-CSF levels in ILS-infected rats. By contrast, upregulation of M-CSF was more robust in WS-infected rats. The phagocytic activity in macrophages harvested from ILS-infected rat spleens, but not WS-infected spleens, was higher than that in macrophages from uninfected rats. These results suggest that macrophages of WS-infected rats change to an immunosuppressive type. However, when WS or ILS is cocultured with spleen macrophages or HS-P cells, a cell line of rat macrophage origin, M-CSF is upregulated relative to GM-CSF and MIF in both cell types. Anemia occurs in ILS-, but not WS-infected, rats. Treatment of spleen macrophages or HS-P cells cocultured with ILS with cobalt chloride, which mimics the effects of anemia-induced hypoxia, led to downregulation of M-CSF mRNA levels, upregulation of GM-CSF and MIF, and an increase in phagocytic activity. However, the effect of cobalt chloride on spleen macrophages and HS-P cells cocultured with WS was restricted. These results suggest that anemia-induced hypoxia in ILS-infected rats stimulates the immune system and activates macrophages.     

Nonvariant antigens limited to bloodstream forms of Trypanosoma brucei brucei and Trypanosoma brucei rhodesiense

The presence of nonvariant antigens (NVAs) limited to bloodstream forms of Trypanosoma brucei brucei and Trypanosoma brucei rhodesiense was demonstrated for the first time by immunodiffusion and immunoelectrophoresis. Noncloned and cloned populations were employed in preparation of polyclonal antisera in rabbits and of antigens to be used in the immunologic reactions. The NVAs could be shown best in systems in which hyperimmune rabbit sera (adsorbed with procyclic forms to eliminate antibodies against antigens common to bloodstream form and procyclic stages) were reacted with trypanosomes characterized by heterologous variant-specific antigens (VSAs). The NVAs demonstrated in this study are very likely different from the common parts of VSAs. As has been suggested by experiments with living trypanosomes, at least a part of the NVAs appears to be located on the surface of the bloodstream forms. In these experiments involving the quantitative indirect fluorescent antibody test, the amount of fluorescence recorded for the heterologous system, i.e. ETat 5 trypanosomes incubated with anti-AmTat 1.1 serum, equalled approximately 3.0% of the fluorescence emitted by the AmTat 1.1 bloodstream forms treated with their homologous antiserum. Evidently, only small amounts of NVAs are present on the surfaces of T. brucei bloodstream forms. In addition to the NVAs, the electrophoresis results suggested the presence of antigenic differences between procyclic stages belonging to different T. brucei stocks.     

Structure of thioredoxin from Trypanosoma brucei brucei

The three-dimensional structure of thioredoxin from Trypanosoma brucei brucei has been determined at 1.4 A resolution. The overall structure is more similar to that of human thioredoxin than to any other thioredoxin structure. The most striking difference to other thioredoxins is the absence of a buried carboxylate behind the active site cysteines. Instead of the common Asp, there is a Trp that binds an ordered water molecule probably involved in the protonation/deprotonation of the more buried cysteine during catalysis. The conserved Trp in the WCGPC sequence motif has an exposed position that can interact with target proteins.     

Transport of alpha-aminoisobutyrate into Trypanosoma brucei brucei

The uptake of alpha-aminoisobutyrate (AIB) by washed cell suspensions of bloodstream forms of Trypanosoma brucei brucei has been shown to be an energy-dependent process. No metabolism of AIB was detected under conditions leading to a 100-fold accumulation of AIB within the organism. Kinetic studies revealed that AIB uptake involved two components; that operating at low substrate concentrations had an apparent Km of 4.6 mM. Experiments with ionophores such as gramicidin and carbonylcyanide m-chlorophenylhydrazone were consistent with the AIB uptake system operating as a H+-symporter responding to the electrochemical gradient of H+, the major component of which was the membrane potential.     

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.     

Trypanosoma brucei brucei: In Vitro Production of Metacyclic Forms

ABSTRACT 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° 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° 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.     

Nonvariant Antigens Limited to Bloodstream Forms of Trypanosoma brucei brucei and Trypanosoma brucei rhodesiense1

ABSTRACT. The presence of nonvariant antigens (NVAs) limited to bloodstream forms of Trypanosoma brucei brucei and Trypanosoma brucei rhodesiense was demonstrated for the first time by immunodiffusion and Immunoelectrophoresis. Noncloned and cloned populations were employed in preparation of polyclonal antisera in rabbits and of antigens to be used in the immunologic reactions. The NVAs could be shown best in systems in which hyperimmune rabbit sera (adsorbed with procyclic forms to eliminate antibodies against antigens common to bloodstream form and procyclic stages) were reacted with trypanosomes characterized by heterologous variant-specific antigens (VSAs). The NVAs demonstrated in this study are very likely different from the common parts of VSAs. As has been suggested by experiments with living trypanosomes, at least a part of the NVAs appears to be located on the surface of the bloodstream forms. In these experiments involving the quantitative indirect fluorescent antibody test, the amount of fluorescence recorded for the heterologous system, i.e. ETat 5 trypanosomes incubated with anti-AmTat 1.1 serum, equalled ～3.0% of the fluorescence emitted by the AmTat 1.1 bloodstream forms treated with their homologous antiserum. Evidently, only small amounts of NVAs are present on the surfaces of T. brucei bloodstream forms. In addition to the NVAs, the electrophoresis results suggested the presence of antigenic differences between procyclic stages belonging to different T. brucei stocks.     

Kinetic characterization, structure modelling studies and crystallization of Trypanosoma brucei enolase.

In this article, we report the results of an analysis of the glycolytic enzyme enolase (2-phospho-d-glycerate hydrolase) of Trypanosoma brucei. Enolase activity was detected in both bloodstream-form and procyclic insect-stage trypanosomes, although a 4.5-fold lower specific activity was found in the cultured procyclic homogenate. Subcellular localization analysis showed that the enzyme is only present in the cytosol. The T. brucei enolase was expressed in Escherichia coli and purified to homogeneity. The kinetic properties of the bacterially expressed enzyme showed strong similarity to those values found for the natural T. brucei enolase present in a cytosolic cell fraction, indicating a proper folding of the enzyme in E. coli. The kinetic properties of T. brucei enolase were also studied in comparison with enolase from rabbit muscle and Saccharomyces cerevisiae. Functionally, similarities were found to exist between the three enzymes: the Michaelis constant (Km) and KA values for the substrates and Mg2+ are very similar. Differences in pH optima for activity, inhibition by excess Mg2+ and susceptibilities to monovalent ions showed that the T. brucei enolase behaves more like the yeast enzyme. Alignment of the amino acid sequences of T. brucei enolase and other eukaryotic and prokaryotic enolases showed that most residues involved in the binding of its ligands are well conserved. Structure modelling of the T. brucei enzyme using the available S. cerevisiae structures as templates indicated that there are some atypical residues (one Lys and two Cys) close to the T. brucei active site. As these residues are absent from the human host enolase and are therefore potentially interesting for drug design, we initiated attempts to determine the three-dimensional structure. T. brucei enolase crystals diffracting at 2.3 A resolution were obtained and will permit us to pursue the determination of structure.