Kinetic studies on the reaction catalysed by phosphofructokinase from Trypanosoma brucei.

The steady-state kinetics of the reaction catalysed by the bloodstream form of Trypanosoma brucei were studied at pH 6.7. In the presence of 50 mM-potassium phosphate buffer, the apparent co-operativity with respect to fructose 6-phosphate and the non-linear relationship between initial velocity and enzyme concentration, which were found when the enzyme was assayed in 50 mM-imidazole buffer [Cronin & Tipton (1985) Biochem. J. 227, 113-124], are not evident. Studies on the variations of the initial rate with changing concentrations of MgATP and fructose 6-phosphate, the product inhibition by fructose 1,6-bisphosphate and the effects of the alternative substrate ITP were consistent with an ordered reaction pathway, in which MgATP binds to the enzyme before fructose 6-phosphate, and fructose 1,6-bisphosphate is the first product to dissociate from the ternary complex.     

Purification and regulatory properties of phosphofructokinase from Trypanosoma (Trypanozoon) brucei brucei.

Phosphofructokinase (EC 2.7.1.11) from Trypanosoma (Trypanozoon) brucei brucei was purified to homogeneity by using a three-step procedure that may be performed within 1 day. Proteolysis, which removes a fragment of Mr approx. 2000, may occur during the purification, but this can be prevented by including antipain, an inhibitor of cysteine proteinases, in the buffers during the purification. The subunits of the enzyme appear to be identical in size, with an Mr of 49 000. The Mr of the native enzyme was estimated to be approx. 220 000, suggesting a tetrameric structure. Kinetic studies showed the activity to depend hyperbolically on the concentration of ATP but sigmoidally on the concentration of fructose 6-phosphate. Although cyclic AMP, AMP and ADP stimulated the enzyme activity at low concentrations of fructose 6-phosphate, the last two nucleotides were inhibitory at high concentrations of this substrate. Phosphoenolpyruvate behaved as an allosteric inhibitor of the phosphofructokinase. Citrate, fructose 1,6-bisphosphate, fructose 2,6-bisphosphate and Pi did not influence significantly the activity of the enzyme.     

The trans-sialidase from the african trypanosome Trypanosoma brucei.

Trypanosoma brucei is the cause of the diseases known as sleeping sickness in humans (T. brucei ssp. gambiense and ssp. rhodesiense) and ngana in domestic animals (T. brucei brucei) in Africa. Procyclic trypomastigotes, the tsetse vector stage, express a surface-bound trans-sialidase that transfers sialic acid to the glycosylphosphatidylinositol anchor of procyclin, a surface glycoprotein covering the parasite surface. Trans-sialidase is a unique enzyme expressed by a few trypanosomatids that allows them to scavenge sialic acid from sialylated compounds present in the infected host. The only enzyme extensively characterized is that of the American trypanosome T. cruzi (TcTS). In this work we identified and characterized the gene encoding the trans-sialidase from T. brucei brucei (TbTS). TbTS genes are present at a small copy number, at variance with American trypanosomes where a large gene family is present. The recombinant TbTS protein has both sialidase and trans-sialidase activity, but it is about 10 times more efficient in transferring than in hydrolysing sialic acid. Its N-terminus contains a region of 372 amino acids that is 45% identical to the catalytic domain of TcTS and contains the relevant residues required for catalysis. The enzymatic activity of mutants at key positions involved in the transfer reaction revealed that the catalytic sites of TcTS and TbTS are likely to be similar, but are not identical. As in the case of TcTS and TrSA, the substitution of a conserved tryptophanyl residue changed the substrate specificity rendering a mutant protein capable of hydrolysing both alpha-(2,3) and alpha-(2,6)-linked sialoconjugates.     

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.     

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.     

Exploring the active site of Trypanosoma brucei phosphofructokinase by inhibition studies: specific irreversible inhibition

This work deals with the phosphofructokinase enzyme (PFK) of the parasite Trypanosoma brucei. Inhibitors which are analogues of fructose-6-phosphate (F6P) derived from 2,5-anhydromannitol and therefore blocked in a closed conformation, both nonphosphorylated and phosphorylated, were designed. They provided information on this class of ATP-dependent PFK (structurally more similar to PPi-dependent PFKs revealing (i) an ordered mechanism, ATP binding first, inducing an essential conformational change to increase the affinity for F6P, and (ii) a rather hydrophobic environment at the ATP binding site. Nonphosphorylated mannitol derivatives bind at both the ATP and F6P binding sites, whereas the phosphorylated derivatives only bind at the ATP binding site. The inhibitors bearing an aromatic ring substituted at the meta position indicate a polar interaction with lysine 227, which is specific to T. brucei PFK and is replaced by a glycine in human PFK. This lysine can be irreversibly bound, leading to inhibition when an electrophilic carbon atom is beta to the meta position on the ring. This lysine was identified by site-directed mutagenesis. This first example of a specific irreversible inactivation of T. brucei PFK offers an opportunity to develop biologically active compounds against the sleeping sickness, the causative agent of which is the trypanosome.     

Identification of coding sequences from a freshly prepared Trypanosoma brucei brucei expression library by polymerase chain reaction

Animal African trypanosomiasis (AAT) also known as Nagana is a devastating disease among domestic animals in large parts of Sub-Saharan Africa causing loses in milk and meat production as well as traction power. However, there is currently no commercial vaccine against AAT. The parasites have also developed resistance to some of the drugs in use. Moreover, the use of affordable computer-aided wet bench methods in the search for vaccine and/or new drug targets against this disease have not yet been fully explored in developing countries. This study, therefore, explored the use of PCR to screen a freshly prepared bloodstream form Trypanosoma brucei brucei (T. b. brucei) expression library for coding sequences followed by bioinformatics analyses specifying the functions and importance of these proteins to parasite survival. Eleven protein coding sequences were identified from twenty nine purified clones. The putative retro transposon hot spot protein 4 (RHSP 4) was the only protein with a fully annotated DNA sequence. All the others were hypothetical or had partial or unqualified annotations. RHSP 4 and pyruvate dehydrogenase E1 component, alpha sub-unit (PDE1α) are involved in aerobic respiration whereas succinyl-Co A-3-ketoacid-coenzyme A transferase mitochondrial precursor (SKTMP) is predicted to be involved in ketone body catabolism. Cystathionine beta-synthase (CBS) and alpha-1,3-mannosyltransferase (αMT) have been predicted in cysteine biosynthesis and vesicular transport respectively. The functions of the hypothetical proteins encountered have neither been experimentally determined nor predicted. We hypothesize that both CBS and PDE1α are good drug targets. Overall, about 300 plates are required to PCR screen the entire Trypanosoma brucei genome in approximately eight months. This method is therefore, applicable and affordable in the search for new drug targets under conditions of limited resources among developing countries.     

The exosome of Trypanosoma brucei.

The yeast exosome is a complex of at least 10 essential 3'-5' riboexonucleases which is involved in 3'-processing of many RNA species. An exosome-like complex has been found or predicted to exist in other eukaryotes but not in Escherichia coli. The unicellular parasite Trypanosoma brucei diverged very early in eukaryotic evolution. We show here that T.brucei contains at least eight exosome subunit homologs, but only a subset of these associate in a complex. Accordingly, the T.brucei exosome is smaller than that of yeast. Both free and complex-associated homologs are essential for cell viability and are involved in 5.8S rRNA maturation. We suggest that the exosome was present in primitive eukaryotes, and became increasingly complex during subsequent evolution.     

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.     

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.     

Purification and assembly in vitro of tubulin from Trypanosoma brucei brucei.

Trypanosome tubulin was purified to near homogeneity by chromatography on DEAE-Sephadex, Amicon filtration and assembly-disassembly in vitro. Polymerization of the tubulin in vitro yielded long, structurally normal, microtubules and some sheet structures on addition of GTP and incubation at 37 degrees C, in either the presence or the absence of Mg2+. Tubulin assembly was disrupted by glycerol and a selection of microtubule-reactive drugs. Immunological analysis of the purified tubulin revealed tyrosinated and acetylated alpha-tubulin, in addition to defining the migration characteristics of the alpha- and beta-tubulin on one-dimensional SDS/polyacrylamide gels. This is the first isolation of trypanosome tubulin with the ability to form structurally normal microtubules independent of the addition of taxol or nucleating microtubule fragments. The development of the purification procedure thus provides an important step for subsequent study of microtubule-associated protein-tubulin and plasma-membrane-microtubule cytoskeleton interactions of trypanosomes, and increases the potential for development of tubulin-based anti-trypanosome drugs.     

The mitochondrion in bloodstream forms of Trypanosoma brucei is energized by the electrogenic pumping of protons catalysed by the F1F0-ATPase.

Bloodstream forms of Trypanosoma brucei were found to maintain a significant membrane potential across their mitochondrial inner membrane (delta psi m) in addition to a plasma membrane potential (delta psi p). Significantly, the delta psi m was selectively abolished by low concentrations of specific inhibitors of the F1F0-ATPase, such as oligomycin, whereas inhibition of mitochondrial respiration with salicylhydroxamic acid was without effect. Thus, the mitochondrial membrane potential is generated and maintained exclusively by the electrogenic translocation of H+, catalysed by the mitochondrial F1F0-ATPase at the expense of ATP rather than by the mitochondrial electron-transport chain present in T. brucei. Consequently, bloodstream forms of T. brucei cannot engage in oxidative phosphorylation. The mitochondrial membrane potential generated by the mitochondrial F1F0-ATPase in intact trypanosomes was calculated after solving the two-compartment problem for the uptake of the lipophilic cation, methyltriphenylphosphonium (MePh3P+) and was shown to have a value of approximately 150 mV. When the value for the delta psi m is combined with that for the mitochondrial pH gradient (Nolan and Voorheis, 1990), the mitochondrial proton-motive force was calculated to be greater than 190 mV. It seems likely that this mitochondrial proton-motive force serves a role in the directional transport of ions and metabolites across the promitochondrial inner membrane during the bloodstream stage of the life cycle, as well as promoting the import of nuclear-encoded protein into the promitochondrion during the transformation of bloodstream forms into the next stage of the life cycle of T. brucei.     

Purification of sn-glycerol-3-phosphate dehydrogenase from Trypanosoma brucei brucei.

A protein has been purified from the membranes of bloodstream forms of Trypanosoma brucei brucei. The purified material contained a single polypeptide chain of molecular mass 67 kilodaltons as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; under "native" conditions it migrated through a Sephacryl S-300 column with a similar molecular mass. The purified protein catalysed electron transfer from sn-glycerol 3-phosphate to oxygen with the subsequent formation of water. Electron transfer by the purified enzyme to O2 was dependent on the presence of low concentrations of the mediator phenazine methosulfate. This protein is clearly the major membrane-bound sn-glycerol-3-phosphate dehydrogenase, but it also has some characteristics suggestive of the trypanosome alternative oxidase activities.     

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.     

Trypanosoma brucei: some properties of the cytotoxic reaction induced by normal human serum.

The trypanocidal activity of normal human serum has been studied in vitro using Trypanosoma brucei as the test organism. The variables affecting the rate and extent of lysis, such as time, temperature, serum concentration, and pleomorphism of trypanosomes, are described. Trypanocidal titers of serum and serum fractions were quantitatively determined under standardized incubation conditions. Inactivation and/or removal of components of both the classical and alternate pathways of complement activation had no effect on the trypanocidal properties of human serum. The active factor was nondialyzable, present in plasma at equivalent levels to that in serum, and not removed by absorption with IgG fractions of antisera against human IgM or α₂-macroglobulin. The trypanocidal factor could be inactivated by heat (65 C), dithiothreitol, urea, and trypsin. Gel filtration studies indicated that the trypanocidal activity eluted as a single protein with a molecular weight of about 500,000.     

The secondary structure of guide RNA molecules from Trypanosoma brucei.

RNA editing in kinetoplastid organisms is a mitochondrial RNA processing phenomenon that is characterized by the insertion and deletion of uridine nucleotides into incomplete mRNAs. Key molecules in the process are guide RNAs which direct the editing reaction by virtue of their primary sequences in an RNA-RNA interaction with the pre-edited mRNAs. To understand the molecular details of this reaction, especially potential RNA folding and unfolding processes as well as assembly phenomena with mitochondrial proteins, we analyzed the secondary structure of four different guide RNAs from Trypanosoma brucei at physiological conditions. By using structure-sensitive chemical and enzymatic probes in combination with spectroscopic techniques we found that the four molecules despite their different primary sequences, fold into similar structures consisting of two imperfect hairpin loops of low thermodynamic stability. The molecules melt in two-state monomolecular transitions with Tms between 33 and 39 degrees C and transition enthalpies of -32 to -38 kcal/mol. Both terminal ends of the RNAs are single-stranded with the 3' ends possibly adopting a single-stranded, helical conformation. Thus, it appears that the gRNA structures are fine tuned to minimize stability for an optimal annealing reaction to the pre-mRNAs while at the same time maximizing higher order structural features to permit the assembly with other mitochondrial components into the editing machinery.