No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Metabolic compartmentation in Trypanosomes
Abstract
Differences between host and parasite energy metabolism are eagerly sought after as potential targets for antiparasite chemotherapy. In Kinetoplastia, the first seven steps of glycolysis are compartmented inside glycosomes, organelles that are related to the peroxisomes of higher eukaryotes. This arrangement is unique in the living world. In this review, Christine Clayton and Paul Michels discuss the implications of this unusual metabolic compartmentation for the regulation of trypanosome energy metabolism, and describe how an adequate supply of energy is maintained in different species and life cycle stages.
... (2011) and Clayton & Michels (1996) about the intense glycolytic pathway activity of bloodstream forms, as previously mentioned [45,46]. ...
... (2011) and Clayton & Michels (1996) about the intense glycolytic pathway activity of bloodstream forms, as previously mentioned [45,46]. ...
... Interestingly, five enzymes involved in energetic metabolism were also observed in the top list. Pyruvate phosphate dikinase, enolase, hexokinase and fructose-bisphosphate aldolase are enzymes involved in highly active glycolysis/gluconeogenesis pathways in T.brucei and T. cruzi bloodstream trypomastigotes, which corroborates previous suggestions that the bloodstream form is more dependent on glycolysis compared to the insect form[45,46]. However, aconitase (citric acid cycle enzyme) has been poorly studied in T. cruzi, and further experiments about its function in the infective parasite form must be performed.Clathrin is a key protein in receptor-mediated endocytosis, promoting the entry of important molecules into eukaryotic cells, including T. cruzi epimastigotes, that present a high endocytic capacity. ...
Unlabelled:
Chagas disease is a neglected disease, caused by the protozoan Trypanosoma cruzi. This kinetoplastid presents a cycle involving different forms and hosts, being trypomastigotes the main infective form. Despite various T. cruzi proteomic studies, the assessment of bloodstream trypomastigote profile remains unexplored. The aim of this work is T. cruzi bloodstream form proteomic description. Employing shotgun approach, 17,394 peptides were identified, corresponding to 7514 proteins of which 5901 belong to T. cruzi. Cytoskeletal proteins, chaperones, bioenergetics-related enzymes, and trans-sialidases are among the top-scoring. GO analysis revealed that all T. cruzi compartments were assessed; and majority of proteins are involved in metabolic processes and/or presented catalytic activity. The comparative analysis between the bloodstream trypomastigotes and cultured-derived or metacyclic trypomastigote proteomic profiles pointed to 2202 proteins exclusively detected in the bloodstream form. These exclusive proteins are related to: (a) surface proteins; (b) non-classical secretion pathway; (c) cytoskeletal dynamics; (d) cell cycle and transcription; (e) proteolysis; (f) redox metabolism; (g) biosynthetic pathways; (h) bioenergetics; (i) protein folding; (j) cell signaling; (k) vesicular traffic; (l) DNA repair; and (m) cell death. This large-scale evaluation of bloodstream trypomastigotes, responsible for the parasite dissemination in the patient, marks a step forward in the comprehension of Chagas disease pathogenesis.
Biological significance:
The hemoflagellate protozoan T. cruzi is the etiological agent of Chagas disease and affects people by the millions in Latin America and other non-endemic countries. The absence of efficient drugs, especially for treatment during the chronic phase of the disease, stimulates the continuous search for novel molecular targets. The identification of essential molecules, particularly those found in clinically relevant forms of the parasite, could be crucial. Inside the vertebrate host, trypomastigotes circulate in the bloodstream before infecting various tissues. The exposure of bloodstream forms of the parasite to the host immune system likely leads to differential protein expression in the parasite. In this context, an extensive characterization of the proteomic profile of bloodstream trypomastigotes could help to find not only promising drug targets but also antigens for vaccines or diagnostics. This work is a large-scale proteomic assessment of bloodstream trypomastigotes that show a considerable number of proteins belonging to different metabolic pathways and functions exclusive to this parasitic form, and provides a valuable dataset for the biological understanding of this clinically relevant form of T. cruzi.
... In particular, the case argued for an endosymbiotic origin of glycosomes (Cavalier-Smith 1997) deserves inspection. Glycosomes are highly specialized forms of peroxisomes that contain enzymes of glycolysis; they occur in trypanosomes (the agents of sleeping sickness) and related organisms (Clayton & Michels 1996). Glycosomes import glucose from the cytosol and export phosphoenolpyruvate and, under some conditions, glycerol or 1,3-bisphosphoglycerate as well (Clayton & Michels 1996). ...
... Glycosomes are highly specialized forms of peroxisomes that contain enzymes of glycolysis; they occur in trypanosomes (the agents of sleeping sickness) and related organisms (Clayton & Michels 1996). Glycosomes import glucose from the cytosol and export phosphoenolpyruvate and, under some conditions, glycerol or 1,3-bisphosphoglycerate as well (Clayton & Michels 1996). The biochemically specialized function of these organelles is twofold. ...
... The biochemically specialized function of these organelles is twofold. They permit e¤cient glycolytic £ux in the bloodstream forms of these parasites (Clayton & Michels 1996;Blattner et al. 1998) but, more importantly, because glycolytic enzymes in trypanosomes are not allosterically regulated, they permit the parasite to adjust its ATP production (and, hence, its growth rate) to the bloodstream glucose concentrations of its host (Bakker et al. 1997). No prokaryotes are known that lack allosteric regulation in their glycolytic pathway (although some are known that lack glycolysis altogether; Andersson et al. 1998). ...
Recent findings are summarized in support of the view that mitochondria (including hydrogenosomes) and plastids (including complex ones) descend from symbiotic associations of once free-living organisms. The reasoning behind endosymbiotic hypotheses stems from a comparison of biochemistry and physiology in organelles with that in free-living cells; their strength is shown to lie in the specific testable predictions they generate about expected similarity patterns among genes. Although disdained for many decades, endosymbiotic hypotheses have gradually become very popular. In the wake of that popularity, endosymbiotic hypotheses have been formulated to explain the origins of eukaryotic cell compartments and structures that have no biochemical similarity to free-living cells. In particular, it has become fashionable in recent years to entertain the century-old notion that the nucleus might also descend from an endosymbiotic bacterium. A critique of that hypothesis is formulated and a simple alternative to it is outlined, which derives the nuclear compartment in a mitochondrion-bearing cell.
... The energy (ATP) metabolism pathway of bloodstream forms (BSFs) of the trypanosomes presents such a novel target for rational drug discovery because it differs from that of animals. Unlike the mammalian host where ATP (cellular energy currency) is produced from multiple pathways such as the glycolysis, citric acid cycle, and oxidative phosphorylation, the BSFs of African trypanosomes rely solely on the metabolism of glucose, i.e., glycolysis, for their energy needs (15,16). The functions of 2 enzymes, the trypanosomal alternative oxidase (TAO) and trypanosomal glycerol kinase (TGK), are critical to the ATP production pathway in BSFs (Supplemental Fig. S1). ...
... A key objective of our study was to discover TbgGK inhibitors that could replace glycerol for combination therapy with AF. Therefore, the effect of the TbgGK inhibitors identified above on the trypanocidal potency of AF was evaluated by the addition of 10 mM each of the secondary hit compounds (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17) at varying AF concentrations on T. b. brucei cultures. The trypanocidal EC 50 (concentration of drug that killed half of the parasite population) of AF was determined. ...
African trypanosomiasis, sleeping sickness in humans or nagana in animals, is a potentially fatal neglected tropical disease and a threat to 65 million human lives and 100 million small and large livestock animals in sub‐Saharan Africa. Available treatments for this devastating disease are few and have limited efficacy, prompting the search for new drug candidates. Simultaneous inhibition of the trypanosomal glycerol kinase (TGK) and trypanosom alalternative oxidase (TAO) is considered a validated strategy toward the development of new drugs. Our goal is to develop a TGK‐specific inhibitor for coadministration with ascofuranone (AF), the most potent TAO inhibitor. Here, we report on the identification of novel compounds with inhibitory potency against TGK. Importantly, one of these compounds (compound 17) and its derivatives (17a and 17b) killed trypanosomes even in the absence of AF. Inhibition kinetics revealed that derivative 17b is a mixed‐type and competitive inhibitor for TGK and TAO, respectively. Structural data revealed the molecular basis of this dual inhibitory action, which, in our opinion, will aid in the successful development of a promising drug to treat trypanosomiasis. Although the EC50 of compound 17b against trypanosome cells was 1.77 μM, it had no effect on cultured human cells, even at 50 μM.—Balogun, E. O., Inaoka, D. K., Shiba, T., Tsuge, C., May, B., Sato, T., Kido, Y., Nara, T., Aoki, T., Honma, T., Tanaka, A., Inoue, M., Matsuoka, S., Michels, P. A. M., Watanabe, Y.‐I., Moore, A. L., Harada, S., Kita, K. Discovery of trypanocidal coumarins with dual inhibition of both the glycerol kinase and alternative oxidase of Trypanosoma brucei brucei. FASEB J. 33, 13002–13013 (2019). www.fasebj.org
... Triosephosphate isomerase (E.C. 5.3.1.1) catalyzes the interconversion between glyceraldehyde-3-phosphate and dihydroxyacetone phosphate in the fifth step of the glycolytic pathway [13]. Structurally, TIM from T. brucei (TbTIM) is a homodimeric enzyme, and each monomer consists of 250 residues forming eight parallel β-strands surrounded by eight α-helices, showing the classical TIM barrel folding [10]. ...
... Physicochemical and toxicological properties of TbTIM inactivators.12.23 (s, 1H, CON-H).13 C NMR (DMSO-d 6 ; 100 MHz) δ: 14.3 (double signal), 113.7 (double signal), 115.1 (double signal), 122.5, 126.7, 135.4, ...
Human African Trypanosomiasis (HAT), a disease that provokes 2184 new cases a year in Sub-Saharan Africa, is caused by Trypanosoma brucei. Current treatments are limited, highly toxic, and parasite strains resistant to them are emerging. Therefore, there is an urgency to find new drugs against HAT. In this context, T. brucei depends on glycolysis as the unique source for ATP supply; therefore, the enzyme triosephosphate isomerase (TIM) is an attractive target for drug design. In the present work, three new benzimidazole derivatives were found as TbTIM inactivators (compounds 1, 2 and 3) with an I50 value of 84, 82 and 73 µM, respectively. Kinetic analyses indicated that the three molecules were selective when tested against human TIM (HsTIM) activity. Additionally, to study their binding mode in TbTIM, we performed a 100 ns molecular dynamics simulation of TbTIM-inactivator complexes. Simulations showed that the binding of compounds disturbs the structure of the protein, affecting the conformations of important domains such as loop 6 and loop 8. In addition, the physicochemical and drug-like parameters showed by the three compounds suggest a good oral absorption. In conclusion, these molecules will serve as a guide to design more potent inactivators that could be used to obtain new drugs against HAT.
... Similarly, T. equiperdum is the only species of African trypanosomes that lives as a tissue parasite (Claes et al, 2005); all other species of develop in the blood and tissue fluids of mammals as free-living organisms that never enter the cells of the host (Pays, 2006). These trypanosomes multiply extracellularly throughout their lifecycle in the blood and tissue fluids of vertebrates and in the alimentary canal of the tsetse fly (Clayton & Michels, 1996). Figure 1 illustrates the relationship between the different species of trypanosomes. ...
... They have also been found to express all the enzymes for gluconeogenesis at this stage (van Hellemond et al, 2005). The epimastigotes of T. cruzi were found to resemble T. brucei procyclics in the use of proline as an important substrate, and in the incomplete catabolism of glucose to CO 2 and organic acids such as alanine and succinate (Clayton & Michels, 1996). The transformation to the metacyclic form is believed to be accompanied by the repression of mitochondrial activity in preparation for infection of the mammalian host (Priest & Hajduk, 1994), since it was found that the metacyclic mitochondrion has the unbranched noncristate appearance of the bloodstream form mitochondrion (Vickerman, 1985). ...
Animal trypanosomiasis is a major hinderance to the growth of livestock farming in sub-Saharan Africa. Chemotherapy using isometamidium, diminazene and ethidium bromide has been the main control method in the absence of a vaccine against this disease. The effectiveness of these few trypanocides is severely threatened by the widespread development of resistance. Therefore, an understanding of the mechanism(s) involved in the development of resistance will assist in the development of screening protocols for easy identification of resistant cases prior to treatment, and also in finding ways to reverse the resistance. We studied the mechanism of resistance to isometamidium in bloodstream forms of Trypanosoma brucei. Resistance to isometamidium in Trypanosoma brucei was found to be composed of a reduced uptake of the drug and the modification of the F1F0 ATPase complex; active drug efflux by ABC transporters was not involved in the resistance mechanism, although efflux of ISM could be observed in both wild-type and resistant lines. Expression of the transporter gene TbAT1, as well as of TbAT-E and TbAT-A, in yeast, each resulted in increased ISM uptake. In addition, the Vmax for the LAPT1 drug transport activity (Low Affinity Pentamidine Transporter) in ISM-resistant trypanosomes (clone ISMR1) was significantly reduced (P<0.05; Student’s t-test) compared to the wild type control. Also, two point mutations, namely G37A and C851A were found in the ATP synthase gamma subunit of the F1F0 ATPase complex of isometamidium-resistant trypanosomes. The resistant clones also lost their mitochondrial DNA and mitochondrial membrane potential and displayed various levels of cross-resistance to ethidium, diminazene, pentamidine and oligomycin. The C851A mutation introduced a stop codon in the open reading frame of the ATP synthase gamma gene. This mutation, when introduced into the wild type Tb427, produced resistance to isometamidium, and cross resistance to diminazene, ethidium, pentamidine and oligomycin. C851A-ATP synthase gamma proves to be a dominant mutation that allows the rapid loss of mitochondrial DNA after just three days exposure of the parasites to 20 nM ISM or ethidium bromide. Finally, following a recent genome-wide loss-of-function RNAi screen that linked TbAQP2 with pentamidine and melarsoprol cross resistance, we were able to demonstrate that TbAQP2 encodes the HAPT1 in T. brucei, thus leaving us with the LAPT1 as the only known T. b. brucei drug transporter of unknown genetic origin. We however identified specific inhibitors for this transporter (LAPT1) that will be of use in its further characterization.
... Immuno-empreintes 1-D de sérums de bovins trypanotolérants (1, 2, 3, 4, 5) et trypanosensibles (6,7,8,9,10) du sécrétome et du protéome d'IL3000. (Clayton et Michels, 1996). ...
... Au sein du glycosome, le NAD+ (Nicotinamide Adénine Dinucléotide, coenzyme d'oxydoréduction) est réduit par la glycérol-3-phosphate déshydrogénase, puis est réoxydé durant la transformation du phosphate dihydroxyacétone en glycérol 3-phosphate, produit principal du métabolisme du glucose dans le glycosome, qui est ensuite transformé en pyruvate dans le cytosol, en aval de la chaine métabolique. Le maintien de l'équilibre ATP/ADP passe par la transformation du glycérol 3-phosphate excédentaire en glycérol par la glycérol kinase(Clayton et Michels, 1996).L'antigène P69 (Boulangé et Authié, 1994) présente une grande homologie avec les protéines BiP (immunoglobulin heavy chain binding protein) des mammifères qui se caractérise par une localisation dans le RE, un rôle de chaperonne et la présence d'une séquence signal hydrophobe situé à l'extrémité N-terminale et d'un tétrapeptide (X) DEL à l'extrémité C-terminale. La divergence entre P69 et les autres BiPs se situe au niveau de cette extrémité C-terminale, que l'on suspecte d'être responsable du caractère hautement immunogène de la protéine. ...
... Trypanosomes, which include disease-causing parasites such as Trypanosoma and Leishmania, offer the best example: In these organisms the seven glycolytic enzymes required to convert glucose into glycerol 3-phosphate and 1,3-bisphosphoglycerate are all contained within a membrane-bound compartment known as the glycosome. This organelle, which is evolutionarily related to the peroxisome, effectively prevents several metabolic intermediates from entering the cytosol (Clayton and Michels 1996). Other research suggested that there may be direct metabolite transfer between glycerol-3-phosphate dehydrogenase and lactate dehydrogenase (Chock and Gutfreund 1988) but that has been contested by later work (Wu, Gutfreund et al. 1991). ...
... Historically, it was believed that compartmentalization of glycolysis in this way increased the speed in which glucose was metabolized in these organisms (Clayton and Michels 1996), but more recent research has shown that the reaction thermodynamics and enzyme kinetics do not support reaction acceleration via channelling in this situation. Instead, the result of glycolysis channelling within the glycosome is hypothesized to be a reduction in cytosolic concentration of glucose-6-phosphate and fructose-1,6-biphosphate. ...
... Trypanosomatid bioenergetics present remarkable differences from mammalian cells, such as the compartmentalisation of several steps of glycolysis into an organelle named the glycosome and mitochondrial ETC differences, accounting for the great majority of reports on T. brucei [45]. Due to their complex life cycles, trypanosomatids adapt to the environment in different hosts, reflecting the functional plasticity of the mitochondrion observed between the parasitic forms [3,41,46,47]. ...
... T. brucei bloodstream forms are essentially glycolytic, living in an environment that presents high glucose levels. In this life stage, many tricarboxylic acid (TCA) cycle enzymes and cytochromes are not expressed in the mitochondrion, affecting energy production [45,50]. However, F 0 -F 1 ATP synthase and consequently the mitochondrial membrane potential (MMP) are still preserved, suggesting basal uncoupled activity in the organelle [51]. ...
The pathogenic trypanosomatids Trypanosoma brucei, Trypanosoma cruzi, and Leishmania spp. are the causative agents of African trypanosomiasis, Chagas disease, and leishmaniasis, respectively. These diseases are considered to be neglected tropical illnesses that persist under conditions of poverty and are concentrated in impoverished populations in the developing world. Novel efficient and nontoxic drugs are urgently needed as substitutes for the currently limited chemotherapy. Trypanosomatids display a single mitochondrion with several peculiar features, such as the presence of different energetic and antioxidant enzymes and a specific arrangement of mitochondrial DNA (kinetoplast DNA). Due to mitochondrial differences between mammals and trypanosomatids, this organelle is an excellent candidate for drug intervention. Additionally, during trypanosomatids' life cycle, the shape and functional plasticity of their single mitochondrion undergo profound alterations, reflecting adaptation to different environments. In an uncoupling situation, the organelle produces high amounts of reactive oxygen species. However, these species role in parasite biology is still controversial, involving parasite death, cell signalling, or even proliferation. Novel perspectives on trypanosomatid-targeting chemotherapy could be developed based on better comprehension of mitochondrial oxidative regulation processes.
... Trypanosomal parasites are equipped with a unique energy metabolism, they live as the bloodstream form in the mammalian host and as the procyclic form in the vector. The procyclic form of T. brucei fulfills its ATP requirement from a cyanide-sensitive and cytochrome-dependent respiratory chain comparable to that observed in the host mitochondria, whereas in the bloodstream form, trypanosomes use the glycolytic pathway, which is localized in a unique organelle the glycosome, as their major source of ATP2345. Once the parasites invade the mammalian host in the bloodstream form, both its cytochrome-dependent respiratory chain and ATP synthesis by oxidative phosphorylation disappear [2,5]. ...
... The procyclic form of T. brucei fulfills its ATP requirement from a cyanide-sensitive and cytochrome-dependent respiratory chain comparable to that observed in the host mitochondria, whereas in the bloodstream form, trypanosomes use the glycolytic pathway, which is localized in a unique organelle the glycosome, as their major source of ATP2345. Once the parasites invade the mammalian host in the bloodstream form, both its cytochrome-dependent respiratory chain and ATP synthesis by oxidative phosphorylation disappear [2,5]. Instead a cyanide-resistant and cytochrome-independent trypanosomal alternative oxidase (TAO) functions as the sole terminal oxidase to re-oxidize NADH accumulated during glycolysis [5]. ...
... The other two mol ATP from glycolysis are produced in the cytosol by the pyruvate kinase (PK) reaction. As a result, equilibrium of ATP consumption and production is established in glycosomes and the glycolytic reaction rate is limited by the glycosomal ATP pool, preventing accumulation of HEB intermediates due to the turbo glycolysis (Bakker et al 2000;Clayton, Michels 1996;Opperdoes 1987;Visser et al 1981). ...
The parasite Trypanosoma brucei is the causative agent of sleeping sickness and involves an insect vector and a mammalian host through its complex life cycle. T. brucei mammalian bloodstream forms (BSF) exhibits unique metabolic features including: (1) reduced expression and activity of mitochondrial enzymes; (2) respiration mediated by the glycerol phosphate shuttle (GPSh) and the Trypanosome alternative oxidase (TAO) that is intrinsically uncoupled from generation of mitochondrial protonmotive force; (3) maintenance of mitochondrial membrane potential by ATP hydrolysis through the reversal of F1FO-ATP synthase activity; (4) strong reliance on glycolysis to meet their energy demands; (5) high susceptibility to oxidants. Here, we critically review the main metabolic features of BSF and provide a hypothesis to explain the unusual metabolic network and its biological significance for this parasite form. We postulate that intrinsically uncoupled respiration provided by the GPSh-TAOsystem acts as a preventive antioxidant defense by limiting mitochondrial superoxide production and complementing the NADPH-dependent scavenging antioxidant defenses to maintain redox balance. Given the uncoupled nature of the GPSh-TAOsystem, BSF avoidscell death processes by maintaining mitochondrial protonmotiveforce through the reversal of ATP synthase activity using the ATP generated by glycolysis. This unique “metabolic design” in BSF has no biological parallel outside of trypanosomatids and highlights the enormous diversity of the parasite mitochondrial processes to adapt to distinct environments.
... The other two ATP molecules from glycolysis are produced in the cytosol by the pyruvate kinase (PK) reaction. As a result, equilibrium of ATP consumption and production is established in glycosomes and the glycolytic reaction rate is limited by the glycosomal ATP pool, preventing accumulation of HEB intermediates due to the turbo glycolysis (Bakker et al 2000;Clayton, Michels 1996;Opperdoes 1987;Visser et al 1981). ...
Theoretical communication Cite Alencar MB, Ramos EV, Silber AM, Zíková A, Oliveira MF (2022) The extraordinary energy metabolism of the bloodstream Trypanosoma brucei forms: a critical review and a hypothesis. Abstract The parasite Trypanosoma brucei is the causative agent of sleeping sickness and involves an insect vector and a mammalian host through its complex life-cycle. T. brucei mammalian bloodstream forms (BSF) exhibits unique metabolic features including: i) reduced expression and activity of mitochondrial enzymes; ii) respiration mediated by the glycerol phosphate shuttle (GPSh) and the Trypanosoma alternative oxidase (TAO) that is intrinsically uncoupled from generation of mitochondrial membrane potential; iii) maintenance of mitochondrial membrane potential by ATP hydrolysis through the reversal of F1Fo ATP synthase activity; iv) strong reliance on glycolysis to meet their
... Par ailleurs, T. brucei présente une grande originalité du métabolisme énergétique, qui est fondamentalement différent selon le stade du cycle parasitaire. Ainsi, si les formes sanguines utilisent le glucose du sang de l'hôte comme source d'énergie, les formes procycliques préfèrent les acides aminés et plus particulièrement la proline, plus abondante chez l'insecte (Clayton et al., 1996). En outre, dans les formes sanguines, le cycle de Krebs et la chaîne respiratoire ne sont pas fonctionnels, la production d'ATP étant alors totalement dépendante de la glycolyse réalisée dans les glycosomes (Bakker et al., 1999), contrairement aux formes insectes. ...
Leishmania et Trypanosoma brucei sont des protozoaires parasites responsables de graves parasitoses de distribution mondiale. Aucun vaccin n'est disponible contre ces maladies dont le traitement reste basé sur un nombre limité de médicaments coûteux, souvent toxiques et peu efficaces, problème auquel s'ajoute celui des chimiorésistances. D'où l'urgente nécessité de trouver de nouvelles cibles pour le développement de nouveaux traitements qui soient à la fois efficaces, non ou moins toxiques et à un coût plus accessible. Les Trypanosomatidés, dont les génomes ont été entièrement séquencés, présentent de nombreuses originalités dans leur biologie cellulaire et moléculaire, par exemple un ADN mitochondrial unique et extrêmement complexe appelé kinétoplaste. Leur développement suit également un "double" cycle cellulaire répliquant, d'une part, classiquement le noyau et, d'autre part, l'ensemble "corps basal-ADN mitochondrial" dont la ségrégation correcte conditionne la cytodiérèse. Ils possèdent par ailleurs deux types de protéasomes, un classique (26S) et un de type procaryote, plus spécifique et absent chez l'homme, le complexe HslVU. Nous avons montré que HslVU est localisé exclusivement dans l'unique mitochondrie des parasites, et qu'il est, chez T. brucei, essentiel pour la survie de ces organismes. En effet, son inhibition par ARN interférence entraine un blocage de la cytodiérèse suivi par une mort cellulaire. Le premier objectif de cette thèse a été de tenter de mieux comprendre le rôle de HslVU dans le cycle cellulaire associé au kinétoplaste chez ces parasites possédant déjà un protéasome classique. Mettant un terme à plusieurs publications contradictoires, nous avons confirmé la localisation mitochondriale de ce complexe chez Leishmania et chez T. brucei. Nous montrons pour la première fois qu'il est tout aussi essentiel dans les formes sanguines, celles présentes chez l'hôte mammifère, que dans les formes procycliques. Nous montrons aussi un rôle différencié des différentes sous-unités du complexe dans le déroulement du cycle cellulaire associé au kinétoplaste. Le deuxième objectif de cette thèse a été d'identifier de nouvelles protéines mitochondriales régulatrices du cycle cellulaire associé au kinétoplaste. Pour ce faire, nous avons développé une approche de criblage par ARN interférence "semi-systématique" sur 104 protéines mitochondriales, principalement de fonction inconnue. Si l'inhibition de l'expression de la majorité de ces protéines (62) n'a aucun effet sur la croissance cellulaire, celle des 42 restantes induit une baisse moyenne ou sévère de cette croissance. De façon surprenante, cette inhibition modifie significativement et avec plus ou moins d'ampleur le déroulement du cycle cellulaire, suggérant qu'il est dépendant de multiples fonctions cellulaires. Finalement, ce travail valide le protéasome HslVU comme une cible thérapeutique pertinente tout particulièrement à l'adresse des formes sanguines de T. brucei. La différenciation fonctionnelle de HslU1 et HslU2 et l'activité indépendante de HslV donnent une image plus complexe sur le fonctionnement de ce protéasome. Les données d'ARN interférence pour leur part nous orientent vers une régulation du cycle cellulaire très intégrée à l'ensemble des activités cellulaires.
... While in the mammalian host, Trypanosoma brucei depends mainly on aerobic metabolism of glucose for ATP generation. The glycosomal triosephosphate isomerase (TIM) is a key enzyme in this metabolic pathway, where it converts dihydroxyacetone phosphate (produced from glucose) to glyceraldehyde 3-phosphate that is utilized in consequent steps to produce ATP [18,37]. ...
The parasite Trypanosoma brucei is the main cause of the sleeping sickness threatening millions of populations in many African countries. The parasitic infection is currently managed by some synthetic medications, most of them suffer limited activity spectrum and/or serious adverse effects. Some studies have pointed out the promising therapeutic potential of the plant extracts rich in polyphenols to curb down parasitic infections caused by T. brucei and other trypanosomes. In this work, the main components dominating Eugenia uniflora and Syzygium samarangense plant extracts were virtually screened, through docking, as inhibitors of seven T. brucei enzymes validated as potential drug targets. The in vitro and in vivo anti-T. brucei activities of the extracts in two treatment doses were evaluated. Moreover, the extract effects on the packed cell volume level, liver, and kidney functions were assessed. Five compounds showed strong docking and minimal binding energy to five target enzymes simultaneously and three other compounds were able to bind strongly to at least four of the target enzymes. These compounds represent lead hits to develop novel trypanocidal agents of natural origin. Both extracts showed moderate in vitro anti-trypanosomal activity. Infected animal groups treated over 5 days with the studied extracts showed an appreciable in vivo anti-trypanosomal activity and ameliorated in a dose dependent manner the anaemia, liver, and kidney damages induced by the infection. In conclusion, Eugenia uniflora and Syzygium samarangense could serve as appealing sources to treat trypanosomes infections.
... TPI (also known as TIM), a ubiquitous enzyme, which is a required enzyme in glycolysis in tumor cells, preferentially selects aerobic glycolysis to gain energy, and the activity of glycolytic metabolism is beneficial to the survival and proliferation of malignancies (Katebi & Jernigan, 2014). TPI catalyzes the isomerization of dihydroxyacetone phosphate (DHAP) to D-glyceraldehyde 3-phosphate (GAP) in the fifth step of the glycolytic pathway because only GAP can be subsequently used for glycolysis-derived ATP synthesis (Clayton & Michels, 1996). TPI is also responsible for obtaining net ATP from glycolysis and producing an extra pyruvate molecule for each glucose molecule, under aerobic and anaerobic conditions (Olivares-Illana et al., 2017). ...
Colorectal cancer (CRC) is a heterogeneous group of diseases that are the result of abnormal glucose metabolism alterations with high lactate production by pyruvate to lactate conversion, which remodels acidosis and offers an evolutional advantage for tumor cells, even enhancing their aggressive phenotype. This review summarizes recent findings that involve multiple genes, molecules, and downstream signaling in the dysregulated glycolytic pathway, which can allow a tumor to initiate acid byproducts and to progress, thereby resulting in acidosis commonly found in the tumor microenvironment of CRC. Moreover, the relationship between CRC cells and the tumor acidic microenvironment, especially for regulating lactate production and lactate dehydrogenase A levels, is also discussed, as well as comprehensively defining different aspects of glycolytic pathways that affect cancer cell proliferation, invasion, and migration. Furthermore, this review concentrates on glucose metabolism-mediated transduction factors in CRC, which include acid-sensing ion channels, triosephosphate isomerase and key glycolysis-related enzymes that regulate glycolytic metabolites, coupled with the effect on tumor cell glycolysis as well as signaling pathways. In conclusion, glucose metabolism mediated by glycolytic pathways that are integral to tumor acidosis in CRC is demonstrated. Therefore, selective metabolic inhibitors or agents against these targets in glucose metabolism through glycolytic pathways may be clinically useful to regulate the tumor's acidic microenvironment for CRC treatment and to identify specific targets that regulate tumor acidosis through a cancer patient-personalized approach. Furthermore, strategies for modifying the metabolic processes that effectively inhibit cancer cell growth and tumor progression and activate potent anticancer effects may provide more effective antitumor prospects for CRC therapy.
... The inhibition of the chaperones' activity in trypomastigotes constantly subjected to extreme temperature variation and to the host immune response could be a promising unexplored strategy to kill the parasite. At the top of the list of abundant proteins in the bloodstream form are four enzymes from glycolysis/gluconeogenesis pathways (pyruvate phosphate dikinase, enolase, hexokinase and fructose-bisphosphate aldolase), reinforcing the dependence of bloodstream trypomastigotes on glycolysis (Clayton and Michels, 1996;Gonçalves et al., 2011). The citric acid cycle enzyme aconitase is among the most abundant proteins in the bloodstream form; however, it is poorly studied in this protozoa. ...
... 41 For instance, in the insect T. brucei respires and primary gets all its ATP by oxidative phosphorylation, while in the host, glycolysis and substrate-level phosphorylation take over and mitochondrial functions are down regulated. 42,43 It is provocative to think that retrograde transport of tRNAs may play a role in such environmental adaptations. ...
Retrograde transport of tRNAs from the cytoplasm to the nucleus was first described in Saccharomyces cerevisiae and most recently in mammalian systems. Although the function of retrograde transport is not completely clear, it plays a role in the cellular response to changes in nutrient availability. Under low nutrient conditions tRNAs are sent from the cytoplasm to nucleus and presumably remain in storage there until nutrient levels improve. However, in S. cerevisiae tRNA retrograde transport is constitutive and occurs even when nutrient levels are adequate. Constitutive transport is important, at least, for the proper maturation of tRNAPhe, which undergoes cytoplasmic splicing, but requires the action of a nuclear modification enzyme that only acts on a spliced tRNA. A lingering question in retrograde tRNA transport is whether it is relegated to S. cerevisiae and multicellular eukaryotes or alternatively, is a pathway with deeper evolutionary roots. In the early branching eukaryote T. brucei, tRNA splicing, like in yeast, occurs in the cytoplasm. In the present report, we have used a combination of cell fractionation and molecular approaches that show the presence of significant amounts of spliced tRNATyr in the nucleus of T. brucei. Notably, the modification enzyme tRNA-guanine transglycosylase (TGT) localizes to the nucleus and, as shown here, is not able to add queuosine (Q) to an intron-containing tRNA. We suggest that retrograde transport is partly the result of the differential intracellular localization of the splicing machinery (cytoplasmic) and a modification enzyme, TGT (nuclear). These findings expand the evolutionary distribution of retrograde transport mechanisms to include early diverging eukaryotes, while highlighting its importance for queuosine biosynthesis.
... In this work we report for the first time the presence of a nucleoside diphosphate kinase in glycosomes, which is expressed only in the mammalian parasite stage trypomastigote. This could be explained because, in contrast to the insect stage, glycolytic enzymes are very abundant in bloodstream stages of mammalian trypanosomatids, which rely mainly on glycolysis and substrate-level phosphorylation for the generation of energy (Clayton and Michels 1996). In addition, the enzymatic content of glycosomes is rapidly changed during parasite stage differentiation (Michels et al. 2006). ...
Nucleoside diphosphate kinases (NDPK) are key enzymes involved in the intracellular nucleotide maintenance in all living organisms, especially in trypanosomatids which are unable to synthesise purines de novo. Four putative NDPK isoforms were identified in the Trypanosoma cruzi Chagas, 1909 genome but only two of them were characterised so far. In this work, we studied a novel isoform from T. cruzi called TcNDPK3. This enzyme presents an atypical N-terminal extension similar to the DM10 domains. In T. cruzi, DM10 sequences targeted other NDPK isoform (TcNDPK2) to the cytoskeleton, but TcNDPK3 was localised in glycosomes despite lacking a typical peroxisomal targeting signal. In addition, TcNDPK3 was found only in the bloodstream trypomastigotes where glyco-lytic enzymes are very abundant. However, TcNDPK3 mRNA was also detected at lower levels in amastigotes suggesting regulation at protein and mRNA level. Finally, 33 TcNDPK3 gene orthologs were identified in the available kinetoplastid genomes. The characterisation of new glycosomal enzymes provides novel targets for drug development to use in therapies of trypanosomatid associated diseases.
... ATP for their energy requirements. 112 Consequently, a number of enzymes with significant influence on the carbohydrate metabolism have been exploited in antitrypanosomatid drug discovery campaigns. The GAPDH is one of the most notable enzymes exploited in this endeavor through structure-based CADD approaches. ...
Despite tremendous improvement in overall global health heralded by the adoption of the Millennium Declaration in the year 2000, tropical infections remain a major health problem in the developing world. Recent estimates indicate that the major tropical infectious diseases namely malaria, tuberculosis, trypanosomiasis and leishmaniasis, account for more than 2.2 million deaths and a loss of approximately 85 million disability adjusted life years annually. The crucial role of chemotherapy in curtailing the deleterious health and economic impacts of these infections has invigorated the search for new drugs against tropical infectious diseases. The research efforts have involved increased application of computational technologies in mainstream drug discovery programs at the hit identification, hit-to-lead and lead optimization stages. This review article highlights various computer-aided drug discovery approaches that have been utilized in efforts to identify novel antimalarial, antitubercular, antitrypanosomal and antileishmanial agents. Focus is largely on developments over the last 5 years (2010-2014).
... With the exception of Trypanosoma evansi, all species within the Trypanosoma genus contain 50-100 complete or partial maxicircles varying in size from 20 Kbp for T. brucei ssp. to 40 kbp for C. fasciculata [48]. The maxicircles encode mitochondrial genes necessary for development and differentiation in the insect vector [48][49][50][51]. ...
Background
Livestock trypanosomoses, caused by three species of the Trypanozoon subgenus, Trypanosoma brucei brucei, T. evansi and T. equiperdum is widely distributed throughout the world and constitutes an important limitation for the production of animal protein. T. evansi and T. equiperdum are morphologically indistinguishable parasites that evolved from a common ancestor but acquired important biological differences, including host range, mode of transmission, distribution, clinical symptoms and pathogenicity. At a molecular level, T. evansi is characterized by the complete loss of the maxicircles of the kinetoplastic DNA, while T. equiperdum has retained maxicircle fragments similar to those present in T. brucei. T. evansi causes the disease known as Surra, Derrengadera or "mal de cadeiras", while T. equiperdum is the etiological agent of dourine or "mal du coit", characterized by venereal transmission and white patches in the genitalia.
Methods
Nine Venezuelan Trypanosoma spp. isolates, from horse, donkey or capybara were genotyped and classified using microsatellite analyses and maxicircle genes. The variables from the microsatellite data and the Procyclin PE repeats matrices were combined using the Hill-Smith method and compared to a group of T. evansi, T. equiperdum and T. brucei reference strains from South America, Asia and Africa using Coinertia analysis. Four maxicircle genes (cytb, cox1, a6 and nd8) were amplified by PCRfrom TeAp-N/D1 and TeGu-N/D1, the two Venezuelan isolates that grouped with the T. equiperdum STIB841/OVI strain. These maxicircle sequences were analyzed by nucleotide BLAST and aligned toorthologous genes from the Trypanozoon subgenus by MUSCLE tools. Phylogenetic trees were constructed using Maximum Parsimony (MP) and Maximum Likelihood (ML) with the MEGA5.1® software.
Results
We characterized microsatellite markers and Procyclin PE repeats of nine Venezuelan Trypanosoma spp. isolates with various degrees of virulence in a mouse model, and compared them to a panel of T. evansi and T. equiperdum reference strains. Coinertia analysis of the combined repeats and previously reported T. brucei brucei microsatellite genotypes revealed three distinct groups. Seven of the Venezuelan isolates grouped with globally distributed T. evansi strains, while TeAp-N/D1 and TeGu-N/D1 strains clustered in a separate group with the T. equiperdum STIB841/OVI strain isolated in South Africa. A third group included T. brucei brucei, two strains previously classified as T. evansi (GX and TC) and one as T. equiperdum (BoTat-1.1). Four maxicircle genes, Cytochrome b, Cythocrome Oxidase subunit 1, ATP synthase subunit 6 and NADH dehydrogenase subunit 8, were identified in the two Venezuelan strains clustering with the T. equiperdum STIB841/OVI strain. Phylogenetic analysis of the cox1 gene sequences further separated these two Venezuelan T. equiperdum strains: TeAp-N/D1 grouped with T. equiperdum strain STIB818 and T. brucei brucei, and TeGu-N/D1 with the T. equiperdum STIB841/OVI strain.
Conclusion
Based on the Coinertia analysis and maxicircle gene sequence phylogeny, TeAp-N/D1 and TeGu-N/D1 constitute the first confirmed T. equiperdum strains described from Latin America.
... Most of its glycolytic enzymes are localized within membrane bound organelles called glycosomes and pyruvate is released out of the cells as a final product of the glycolysis instead of lactate in mammalian cells. T. brucei bloodstream forms essentially depend on glycolysis as many enzymes of the tricarboxylic acid cycle and cytochromes are not expressed in the mitochondrion [13]. This shows the physiological essentiality of pyruvate export in T. brucei. ...
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.
... The glucose-based metabolism is a key metabolic pathway for bloodstream forms, the mammalian infective stages. The absence of a fully functional mitochondrion along with a remarkable high proliferation rate makes parasites entirely dependent on glucose [4,5]. The glucose-based metabolism comprises two pathways: the glycolytic pathway and the pentose phosphate pathway (PPP). ...
Ribose 5-phosphate isomerase is an enzyme involved in the non-oxidative branch of the pentose phosphate pathway, and catalyzes the inter-conversion of D-ribose 5-phosphate and D-ribulose 5-phosphate. Trypanosomatids, including the agent of African sleeping sickness namely Trypanosoma brucei, have a type B ribose-5-phosphate isomerase. This enzyme is absent from humans, which have a structurally unrelated ribose 5-phosphate isomerase type A, and therefore has been proposed as an attractive drug target waiting further characterization. In this study, Trypanosoma brucei ribose 5-phosphate isomerase B showed in vitro isomerase activity. RNAi against this enzyme reduced parasites' in vitro growth, and more importantly, bloodstream forms infectivity. Mice infected with induced RNAi clones exhibited lower parasitaemia and a prolonged survival compared to control mice. Phenotypic reversion was achieved by complementing induced RNAi clones with an ectopic copy of Trypanosoma cruzi gene. Our results present the first functional characterization of Trypanosoma brucei ribose 5-phosphate isomerase B, and show the relevance of an enzyme belonging to the non-oxidative branch of the pentose phosphate pathway in the context of Trypanosoma brucei infection.
... This is in contrast to the mitochondrial localization of ␣-KDE2 in BF T. brucei (12). The punctate cytoplasmic localization of ␣-KDE1-HA was reminiscent of the distribution of glycosomes in T. brucei (29,30). Immunofluorescence micros- copy with an antibody against the glycolytic enzyme aldolase confirmed that ␣-KDE1-HA was localized to glycosomes in BF T. brucei (Fig. 6C). ...
The α-ketoglutarate decarboxylase (α-KDE1) is a Krebs cycle enzyme found in the mitochondrion of the procyclic form (PF) Trypanosoma brucei. The bloodstream form (BF) of T. brucei lacks a functional Krebs cycle and relies exclusively on glycolysis for ATP production. Despite the lack of a functional Krebs cycle α-KDE1 was expressed in BF T. brucei and RNAi knockdown of α-KDE1 mRNA resulted in rapid growth arrest and killing. Cell death was preceded by progressive swelling of the flagellar pocket as a consequence of recruitment of both flagella and plasma membranes into the pocket. Bloodstream form T. brucei expressing an epitope tagged copy of α-KDE1 showed localization to glycosomes and not the mitochondrion. We used a cell line transfected with a reporter construct containing the N-terminal sequence of α-KDE1 fused to GFP to examine the requirements for glycosome targeting. We found that the N-terminal 18 amino acids of α-KDE1 contained overlapping mitochondria and peroxisome targeting sequences and was sufficient to direct localization to the glycosome in BF T. brucei. These results suggest that α-KDE1 has a novel moonlighting function outside the mitochondrion in BF T. brucei.
Copyright © 2014, American Society for Microbiology. All Rights Reserved.
... Trypanosomes undergo a complex life cycle, with the procyclic form (PF) inhabiting the gut of a tsetse fly and the bloodstream form (BF) being pathogenic in vertebrate hosts. The cyclic changes between the BF and PF are accompanied by dramatic changes in the parasite's metabolism, particularly by the activation of the citric acid cycle and respiratory chain in the mitochondrion of the PF and their downregulation, followed by a switch to glycolysis in the BF (Clayton and Michels 1996; Besteiro et al. 2005). Trypanosoma brucei belongs to the class Kinetoplastea, which commonly have a large network of mitochondrial DNA termed the kinetoplast. ...
Mitochondrial processing peptidase (MPP) consists of α and β subunits that catalyze the cleavage of N-terminal mitochondrial-targeting
sequences (N-MTSs) and deliver preproteins to the mitochondria. In plants, both MPP subunits are associated with the respiratory
complex bc1, which has been proposed to represent an ancestral form. Subsequent duplication of MPP subunits resulted in separate sets
of genes encoding soluble MPP in the matrix and core proteins (cp1 and cp2) of the membrane-embedded bc1 complex. As only α-MPP was duplicated in Neurospora, its single β–MPP functions in both MPP and bc1 complexes. Herein, we investigated the MPP/core protein family and N-MTSs in the kinetoplastid Trypanosoma brucei, which is often considered one of the most ancient eukaryotes. Analysis of N-MTSs predicted in 336 mitochondrial proteins
showed that trypanosomal N-MTSs were comparable with N-MTSs from other organisms. N-MTS cleavage is mediated by a standard
heterodimeric MPP, which is present in the matrix of procyclic and bloodstream trypanosomes, and its expression is essential
for the parasite. Distinct Genes encode cp1 and cp2, and in the bloodstream forms the expression of cp1 is downregulated along
with the bc1 complex. Phylogenetic analysis revealed that all eukaryotic lineages include members with a Neurospora-type MPP/core protein family, whereas cp1 evolved independently in metazoans, some fungi and kinetoplastids. Evolution of
cp1 allowed the independent regulation of respiration and protein import, which is essential for the procyclic and bloodstream
forms of T. brucei. These results indicate that T. brucei possesses a highly derived MPP/core protein family that likely evolved in response to its complex life cycle and does not
appear to have an ancient character proposed earlier for this eukaryote.
... The procyclic forms rely more on mitochondrial activity. Substrates other than glucose, such as amino acids and fatty acids, are then also used [4]. Glycosomes are organelles closely related to peroxisomes of other eukaryotes. ...
Kinetoplastid organisms, such as the protozoan parasite Trypanosoma brucei, compartmentalise several important metabolic pathways in organelles called glycosomes. Glycosomes are related to peroxisomes of yeast and mammalian cells. A subset of glycosomal matrix proteins is routed to the organelles via the peroxisome-targeting signal type 1 (PTS-1). The PEX5 gene homologue has been cloned from T. brucei coding for a protein of the translocation machinery, the PTS-1 receptor. The gene codes for a polypeptide of 654 amino acids with a calculated molecular mass of 70 kDa. Like its homologue in other organisms T. brucei PTS-1 receptor protein (TbPEX5) is a member of the tetratricopeptide repeat (TPR) protein family and contains several copies of the pentapeptide W-X-X-X-F/Y. Northern and Western blot analysis showed that the protein is expressed at different stages of the life cycle of the parasite. The protein has been overproduced in Escherichia coli and purified using immobilized metal affinity chromatography. The purified protein specifically interacts in 6itro with glycosomal phosphoglycerate kinase-C (PGK-C) of T. brucei, a PTS-1 containing protein. The equilibrium dissociation constant (K d) of PGK-C for purified TbPEX5 is 40 nM. Using biochemical and cytochemical techniques a predominantly cytosolic localization was found for TbPEX5. This is consistent with the idea of receptor cycling between the glycosomes and the cytosol. © 1999 Elsevier Science B.V. All rights reserved.
... These diseases are transmitted to mammalian hosts by tsetse fly. During their digenetic life cycle these parasites possess two main stages: the bloodstream form, in the mammalian host, and the procyclic form that lives in the insect vector with changes in metabolism, morphology, and plasma membrane composition (Vickerman, 1985;Clayton and Michels, 1996). The plasma membrane of cells may contain enzymes whose active sites face the external medium. ...
Trypanosoma brucei brucei is the causative agent of animal African trypanosomiasis, also called nagana. Procyclic vector form resides in the midgut of the tsetse fly, which feeds exclusively on blood. Hemoglobin digestion occurs in the midgut resulting in an intense release of free heme. In the present study we show that the magnesium-dependent ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase) activity of procyclic T. brucei brucei is inhibited by ferrous iron and heme. The inhibition of E-NTPDase activity by ferrous iron, but not by heme, was prevented by pre-incubation of cells with catalase. However, antioxidants that permeate cells, such as PEG-catalase and N-acetyl-cysteine prevented the inhibition of E-NTPDase by heme. Ferrous iron was able to induce an increase in lipid peroxidation, while heme did not. Therefore, both ferrous iron and heme can inhibit E-NTPDase activity of T. brucei brucei by means of formation of reactive oxygen species, but apparently acting through distinct mechanisms.
... Due to the initially under-appreciated role of Lproline metabolism, previous reviews concerning energy metabolism in insect trypanosomatid forms focused principally on glucose metabolism (23,80,(91)(92)(93)(94)(95)(96)(97). Given the current understanding that L-proline is the primary carbon source for insect parasite forms, in the present review we describe in detail L-proline metabolism in trypanosomatids. ...
Trypanosomatids are a large family of unicellular eukaryotes, many of which are parasites in higher eukaryotes including man. Much of our understanding of metabolism in these organisms has been gained form the study of the human infective representatives (Trypanosoma brucei subpecies, Trypanosoma cruzi and Leishmania spp.) which are transmitted by blood-feeding arthropods. The insect vectors of these parasites use proline as a principal carbon and energy source circulating in their haemolymph. Accordingly the insect-forms of the human infectious parasites have evolved to exploit abundant proline when in this environment, but being able to activate different biochemical pathways when in other environments. Interestingly, if glucose is available, metabolic capability can shift to make this carbohydrate the preferred substrate. Proline has also been shown to play key roles in osmoregulation, differentiation in representatives of the group and may even play a role in immunosuppression elicited by the American trypanosome T. cruzi. This review focuses on recent progress in understanding the different aspects of proline metabolism in trypanosomatids, with a particular interest on the insect forms.
... La excreción al medio de cultivo de estos ácidos orgánicos como metabolitos finales hace que el pH del medio baje (datos nos mostrados). La cepa de referencia TCI presenta el metabolismo glucolítico muy semejante a los que presentan las formas procíclicas de T. brucei, en donde se produce como degradación parcial de la glucosa, los metabolitos: alanina, acetato y succinato (Clayton y Michel, 1996). La producción de acetato parece ser común a todas las cepas de T. cruzi estudiadas: ...
Tesis Univ. Granada. Instituto de Biotecnología. Leída el 25 de noviembre de 2005
Diseases caused by trypanosomatids include leishmaniasis (Leishmania spp.), Chagas disease (Trypanosoma cruzi), and sleeping sickness (Trypanosoma brucei) that affect millions of people, especially low-income populations, being classified as neglected tropical diseases. Limitations in the clinical treatment, associated with the huge number of cases, make these infections a health and socioeconomic problem worldwide. To complete their life cycle, trypanosomatids survive to environmental changes in different hosts, including oxidative stress. A paradoxal role of reactive oxygen species (ROS) has been proposed, such as signaling as a proliferation regulator or even presenting cytotoxic activity, depending on the concentration. Mitochondrial electron transport chain, especially complex III, is figured as one of the most important ROS resources in trypanosomatids. In relation to antioxidant defenses, trypanothione pathway plays a crucial role, being a peculiar thiol-redox system responsible for the maintenance of protozoa functions mediated by thiol-dependent processes. In this chapter, we discuss the biological aspects of oxidative stress in trypanosomatids and its implications for the success of the infection. The possible ROS resources in these protozoa and their consequent antioxidant machinery involved in detoxification were also focused in this review, including alternative strategies for the development of new drugs for these diseases based on oxidative stress modulation.
Mitosomes are highly reduced forms of mitochondria, which were found in several parasitic protists of various eukaryotic lineages, including the human parasites Entamoeba histolytica, Giardia intestinalis, Cryptosporidium spp., Mikrocytos mackini, and microsporidians. Although all these organisms underwent different evolutionary histories, they arrived at common life strategies for which oxygen-dependent ATP synthesis is not required: they inhabit either an oxygen-poor environment such as the intestinal tract of their hosts or they are adapted to intracellular parasitism. Consequently, the majority of their mitochondrial functions were permanently lost including ATP synthesis with concomitant loss of the organellar genome. The common features of mitosomes, which were retained and pointed to their mitochondrial origin, are a double membrane surrounding the organellar matrix, conserved mechanisms of protein import and processing, and the biosynthesis of iron-sulfur (Fe-S) clusters. Finding the latter function in mitosomes supports the notion that Fe-S cluster assembly is the only essential function of mitochondria necessary for the maturation of cellular Fe-S proteins. Only in the mitosomes of Entamoeba histolytica the mitochondrion type of Fe-S cluster assembly machinery was not conserved, and these organelles gained a unique sulfate activation pathway. In spite of a great progress in elucidation of the evolutionary paths leading to the formation of mitosomes, cellular functions of mitosomes are still poorly understood.
Trypanosoma brucei, the agent of African Trypanosomiasis, is a flagellated protozoan parasite that develops in tsetse flies and in the blood of various mammals. The parasite acquires nutrients such as sugars, lipids and amino acids from their hosts. Amino acids are used to generate energy and for protein and lipid synthesis. However, it is still unknown how T. brucei catabolizes most of the acquired amino acids. Here we explored the role of an enzyme of the leucine catabolism, the 3-methylglutaconyl-Coenzyme A hydratase (3-MGCoA-H). It catalyzes the hydration of 3-methylglutaconyl-Coenzyme A (3-MGCoA) into 3-hydroxymethylglutaryl-Coenzyme A (3-HMGCoA). We found that 3-MGCoA-H localizes in the mitochondrial matrix and is expressed in both insect and mammalian bloodstream forms of the parasite. The depletion of 3-MGCoA-H by RNA interference affected minimally the proliferation of both forms. However, an excess of leucine in the culture medium caused growth defects in cells depleted of 3-MGCoA-H, which could be reestablished by mevalonate, a precursor of isoprenoids and steroids. Indeed, procyclics depleted of the 3-MGCoA-H presented reduced levels of synthesized steroids relative to cholesterol that is scavenged by the parasite, and these levels were also reestablished by mevalonate. These results suggest that accumulation of leucine catabolites could affect the level of mevalonate and consequently inhibit the sterol biosynthesis, required for T. brucei growth.
The mutualistic relationship between trypanosomatids and their respective endosymbiotic bacteria represents an excellent model for studying metabolic co-evolution since the symbiont completes essential biosynthetic routes of the host cell. In this work, we investigated the influence of the endosymbiont on the energy metabolism of Strigomonas culicis by comparing the wild strain with aposymbiotic protists. The bacterium maintains a frequent and close association with glycosomes, which are distributed around the prokaryote. Furthermore, 3D reconstructions revealed that the shape and distribution of glycosomes are different in symbiont-bearing protists compared to symbiont-free cells. Results of bioenergetic assays showed that the presence of the symbiont enhances the O2 consumption of the host cell. When the quantity of intracellular or released glycerol was evaluated, the aposymbiotic strain presented higher values when compared to symbiont-containing cells. Furthermore, inhibition of oxidative phosphorylation by potassium cyanide increased the rate of glycerol release and slightly diminished the ATP content in cells without the symbiont, indicating that the host trypanosomatid enhances its fermentative activity when the bacterium is lost.
Biological systems are governed by their inherent complex molecular regulatory networks at multiple levels. It is the underlying chemical interactions that lead to the observed functions, phenotypic characteristics and diversity. However, it is not clear how such complex interactions at the molecular level impart specific functional characteristics of the cell/organism. This review presents a detailed account on how basic biochemical kinetics principles and computational tools are utilized to mathematically model and analyze such complex regulations. Such approaches are of crucial value in terms of (i) understanding disease mechanisms at molecular level, (ii) to predict possible drug targets (iii) to design new experiments and (iv) to combine the new experimental data along with known information to simulate drug specific pharmaco-kinetic behaviour of the system of investigation which might assist in treatment optimization. This review will also discuss a few case based examples reported in this emerging area to elucidate how such tools are successfully utilized to gain insights about biological systems for medical or drug discovery applications.
This chapter presents an overview of image-based screening methods, highlighting both its advantages and challenges, and presenting examples where this approach has been used to profile natural products libraries for drug discovery. It presents the major classes of organisms that have been used in image-based screening platforms. Selected studies that have used these platforms to engage in NP discovery are reviewed, and several areas where future screening of natural products holds promise for the discovery of new lead compounds are highlighted. Parasites that reside in the bloodstream rather than in invading host cells have traditionally been screened in axenic culture under high-throughput screening (HTS) conditions. In general, this approach is very successful, and is an excellent example of targets for which high-content screening (HCS) offers little advantage over plate reader-based methods, given that phenotypic differentiation is difficult at the magnifications achievable with most well plate-imaging systems. This edition first published 2014
Collectively, diseases caused by parasitic protozoa, including human African trypanosomiasis (HAT), Chagas' disease and leishmaniasis, threaten more than 550 million people worldwide and cause nearly 150,000 deaths annually. The causative organisms for these diseases are unicellular trypanosomatid parasites of the genera Trypanosoma brucei, Trypanosoma cruzi, and Leishmania sp., respectively. Drug therapies available for these diseases have not changed significantly in the past 50 years, and currently used agents are far less than satisfactory due to extreme toxicity, and because resistant parasitic strains are becoming more prevalent. These diseases are confined to impoverished or rural areas of Mexico, Central America, South America, sub-Saharan Africa, the Middle East, Indonesia, and India. As such, drug discovery efforts against trypanosomatid diseases are limited because patients in underdeveloped areas cannot afford therapy, and because the infected population is too small to justify the required research expenditures. In addition, antiparasitic research in Third World nations is often hampered by economic issues and political turmoil, virtually assuring that the world's most impoverished people will continue to bear the major burden of parasitic disease. Clearly, there is a need for new anti-infective agents that are potent, nontoxic and inexpensive to manufacture. In this chapter, the etiology of these diseases and their current treatment are described. The chapter also deals with medicinal chemistry aspects of efforts to identify new drug targets for parasitic diseases, and to produce novel inhibitors of trypanosomatid growth for use as antiparasitic agents.
RNA editing in the parasitic organism Trypanosoma brucei is characterised by the insertion and deletion of uridylate residues into otherwise incomplete primary transcripts. The processing
reaction is a required pathway for the expression of most mitochondrial genes and proceeds by a cascade of enzyme-catalysed
steps. RNA editing involves one or more macromolecular ribonucleoprotein complexes which are likely to interact with additional
components as the reaction proceeds. Here we examined the involvement of the gRNA-binding polypeptide gBP21, a protein which
has been demonstrated to be associated with active RNA editing complexes. We show that in vitro RNA editing can be suppressed by the addition of a gBP21-specific antibody or by immunodepletion of the protein. By creating
a gBP21 knockout mutant we analysed the requirement for the protein in vivo. gBP21− trypanosomes are viable as bloodstream stage cells and contain edited mRNAs. However, the knockout mutant is not capable
of differentiating from the bloodstream to the insect life cycle stage in vitro. Moreover, mutant cells are characterised by a low mitochondrial transcript abundance. Together, these data establish that
gBP21 contributes a non-essential function to the RNA editing reaction and further suggest that the protein is involved in
additional mitochondrial processes which impact a larger pool of mitochondrial transcripts.
Die Übersetzer des genetischen Codes sind in allen Lebewesen die tRNA-Moleküle. Diese 70 bis 80 Nukleotide langen RNA-Moleküle werden zunächst in Bakterien wie in Menschen als lange Vorläufermoleküle synthetisiert. Spezielle Enzyme schneiden dann die tRNA heraus. Während das Enzym RNase P für den Schnitt am vorderen Ende seinen Entdeckern bereits vor mehr als zwanzig Jahren einen Nobelpreis einbrachte, entzog sich das Enzym für das hintere Ende bisher der Charakterisierung. Jetzt gelang Anita Marchfelder und ihrem Team in der Molekularen Botanik der Universität Ulm die Isolierung dieses Proteins.
Leishmania and Trypanosoma belong to the Trypanosomatidae family and cause important human infections such as leishmaniasis, Chagas disease, and sleeping sickness. Leishmaniasis, caused by protozoa belonging to Leishmania, affects about 12 million people worldwide and can present different clinical manifestations, i.e., visceral leishmaniasis (VL), cutaneous leishmaniasis (CL), mucocutaneous leishmaniasis (MCL), diffuse cutaneous leishmaniasis (DCL), and post-kala-azar dermal leishmaniasis (PKDL). Chagas disease, also known as American trypanosomiasis, is caused by Trypanosoma cruzi and is mainly prevalent in Latin America but is increasingly occurring in the United States, Canada, and Europe. Sleeping sickness or human African trypanosomiasis (HAT), caused by two sub-species of Trypanosoma brucei (i.e., T. b. rhodesiense and T. b. gambiense), occurs only in sub-Saharan Africa countries. These pathogenic trypanosomatids alternate between invertebrate and vertebrate hosts throughout their lifecycles, and different developmental stages can live inside the host cells and circulate in the bloodstream or in the insect gut. Trypanosomatids have a classical eukaryotic ultrastructural organization with some of the same main organelles found in mammalian host cells, while also containing special structures and organelles that are absent in other eukaryotic organisms. For example, the mitochondrion is ramified and contains a region known as the kinetoplast, which houses the mitochondrial DNA. Also, the glycosomes are specialized peroxisomes containing glycolytic pathway enzymes. Moreover, a layer of subpellicular microtubules confers mechanic rigidity to the cell. Some of these structures have been investigated to determine their function and identify potential enzymes and metabolic pathways that may constitute targets for new chemotherapeutic drugs.
In Leishmania mexicana two genes were detected coding for different isoforms of the glycolytic enzyme phosphoglycerate kinase. This situation contrasts with that observed in other Trypanosomatidae (Trypanosoma brucei, Trypanosoma congolense, Crithidia fasciculata) analyzed previously, which all contain three different genes coding for isoenzymes A, B and C, respectively. All attempts to detect in L. mexicana a type A PGK, or a gene encoding it, proved unsuccesful. We have cloned and characterized the genes PGKB and PGKC. They code for polypeptides of 416 and 478 amino acids with a molecular mass of 45146 and 51318 Da, respectively. The two polypeptides are 99% identical. PGKC is characterized by a 62 residue C-terminal extension with alternating stretches of hydrophobic and charged, mainly positive amino acids. As in other Trypanosomatidae, PGKB is located in the cytosol, PGKC in the glycosomes. However, Leishmania mexicana distinguishes itself from other trypanosomatids by the simultaneous expression of these isoenzymes: ≈80% of PGK activity is found in the cytosol and 20% in the glycosomes, both in promastigotes and in the amastigote-like form of the parasite.
Five species of copepod crustaceans from four lakes in southern Chile were experimentally exposed to coracidia of Diphyllobothrium latum. Diphyllobothrium latum developed into mature procercoids in the calanoid copepods Boeckella gracilis and Diaptomus diabolicus, but only developed into the initial stage in the cyclopoid copepod Mesocyclops longisetus. The cyclopoid copepods Tropocyclops prasinus and Metacyclops mendocinus were not susceptible to the infection. In our experimental infections we obtained a prevalence of 26.8% in B. gracilis from Lake Malleco, 55.9% in D. diabolicus from Lake Rapel, 84.3% in D. diabolicus from Lake Panguipulli, and 7.7–22.6% for M. longisetus from Lake Panguipulli. The mean and maximum intensity were observed in D. diabolicus from Lake Panguipulli. We conclude that D. diabolicus is probably the primary intermediate host of D. latum because it is the most widespread copepod in southern Chile.
The threat to human health and the developmental constraints imposed by African trypanosomes require continued efforts in epidemiology and drug development, using new approaches and exploiting discoveries in the laboratory. There have been several important advances in the study of trypanosome metabolism, gene regulation and growth control, giving some promise for the development of new approaches to control.
Dinoflagellates are unicellular flagellated eukaryotes exploiting different nutritional modes although approximately half
of them are photosynthetic. They are a monophyletic group, included in the lineage Alveolates. Dinoflagellates are ecologically
important as components of the phytoplankton and contribute significantly to CO2 fixation and primary productivity in the oceans. As well, they can form blooms (densities of more than a million cells per
millilitre) producing red and brown tides. Recently the possible role of programmed cell death (PCD) in phytoplankton has
received much attention because massive cell disappearance of species as a consequence of cell death have important consequences
in the ocean dynamics. Several species of phytoplankters undergo PCD, apparently using the same core mechanism as metazoans.
However, dinoflagellates show different PCD morphologies (apoptotic, necrotic, necrotic-like and paraptotic) depending on
the species and on the triggering factor, probably due to their mesokaryotic condition. Similarly to metazoans, PCD in this
group goes through intermediates such as ROS, and the proteins in charge of executing the cell are metacaspases (cysteinyl
aspartate proteases from the caspase family, found in yeast, plants, protists and some bacteria). The acquisition of the PCD
genes in these organisms goes back to ancient times when the endosymbiotic events took place. However, the intriguing point
is the gene persistence through evolution, given that such genes provide the cell with negative selective pressure.
Since the discovery of programmed cell death in multicellular organisms and due to its definition as a mechanism to maintain
the individual haemostasis of cellular and organ integrity, it was not plausible to think that such a phenomenon could also
occur in unicellular organisms. However, during the last decade considerable experimental evidence has accumulated that confirm
the existence of programmed (i.e., genetically encoded) mechanisms of cell death in a wide variety of single-cell organisms,
including bacteria as well as free living and parasitic protozoa. Moreover, the discovery of biofilm formation and quorum
sensing in bacteria1 and similar observations especially in protozoan parasites2 has changed our perception not to view unicellular organisms as selfish, self-contained and autonomous entities but as well
organized cell populations expressing established communication patterns, thus resembling their multicellular counterparts.
In this chapter we will summarize the most obvious findings regarding programmed cell death in African trypanosomes.
Mitosomes are simple, mitochondrion-derived organelles, which were recently found in various “amitochondrial”
protists, including the human parasites Entamoeba histolytica, Giardia intestinalis,
Cryptosporidium parvum, and microsporidians. Similar organelles might also be present in some
free-living protists. Although all these organisms underwent different evolutionary histories, they arrived
at common life strategies for which oxygen-dependent ATP synthesis is not required: they inhabit either
an oxygen-poor environment, such as the intestinal tract of their hosts, or they are adapted to intracellular
parasitism. Consequently, the majority of their mitochondrial functions were permanently lost with concomitant
loss of the organellar genome, and mitochondria gradually transformed into their highly reduced forms named
mitosomes. The common features of mitosomes, which were retained and pointed to their mitochondrial origin,
are adouble membrane surrounding the organellar matrix, conserved mechanisms of protein import and
processing, and the biosynthesis of iron–sulfur (FeS) clusters. Finding the latter function in mitosomes
supports the notion that FeS cluster assembly is the only essential function of mitochondria necessary for
the maturation of cellular FeS proteins. Only in the mitosomes of E.histolytica
was the mitochondrion type of FeS cluster assembly machinery not conserved, and their function remains enigmatic.
Unlike hydrogenosomes, another type of mitochondrion-derived organelle, mitosomes do not synthesize ATP
and hydrogen. Many more investigations are required to elucidate the biology of mitosomes and the evolutionary
paths leading to the formation of the various mitochondrion-derived organelles, of which mitosomes are the
most simplified.
The chapter provides coverage of all drugs currently used for the treatment of human African trypanosomiasis (African sleeping sickness), American trypanosomiases (Chagas’ disease), and leishmaniasis. Collectively, this group of diseases is caused by flagellated protozoan of the order Kinetoplastida. The drugs currently used to treat these diseases include melarsoprol, suramin, pentamidine, organic antimonials, and several nitroheterocyclic agents. The agents have all been in use for 25–75 years. They have a high degree of toxicity and the first four in the preceding list must be given by parenteral injection over a 2- to 6-week time period. Tolerance has developed to most of the drugs in at least some geographical areas. Eflornithine is the only agent approved in the last 50 years for the treatment of African trypanosomiasis, although it is expensive, requires prolonged systemic dosage, and is effective only against T. brucei gambiense. Megazol, trybazine, DB289, and CGP 40215A have all yielded encouraging results in in vivo preclinical studies against African trypanosomiasis. All four of these agents have entered or are scheduled to enter clinical trials. Nifurtimox and benznidazole are the only agents approved to treat Chagas’ disease. The agents are toxic, rarely produce long-term cures, and resistance to them has developed. The third-generation triazole antifungal agents posaconazole and UR-9825 have given encouraging preclinical results against Chagas’ disease. Meltefosine has been found to be orally active and to produce 98% cure rates of visceral leishmaniasis. New classes of experimental antitrypanosomal agents include the bisphosphonates, which disrupt lipid metabolism, and cysteine protease inhibitors. Agents from both of these mechanistic classes are being advanced as potential antitrypanosomal agents. Kinetoplastid biochemistry offers numerous opportunities for the development of chemotherapeutic agents having a high degree of selective toxicity. Trypanothione, the N¹,N⁸-bis(glutathionyl)spermidine metabolite that replaces glutathione in redox protection reactions in kinetoplastids, is one hopeful target for drug design. A second possible target is the kinetoplastid organelle named the glycosome. African trypanosomes and perhaps other kinetoplastids are highly dependent on glycolysis for energy production. Enclosing the enzymes for glycolysis in glycosomes increases the efficiency of glycolysis compared to its rate in mammalian cells. Any potent inhibitor of an enzyme contained in the glycosome, or of any of the biochemical processes associated with glycosomal function, might be an effective drug against these parasites.
The insect form of Trypanosoma brucei depends on respiration for its energy requirements. It contains a fully functional mitochondrion with a complete citric acid cycle. Most of its enyzmes have been characterized to date. The current study presents the characterization of the histidine phosphorylation activity of one of the few remaining enzymes, succinyl CoA synthetase. The trypanosomal enyzme was identified by partial purification, followed by direct protein sequencing. It is rapidly phosphorylated, presumably through auto-phosphorylation, using either ATP or GTP as phosphate donors. The phosphorylation occurs exclusively on histidine residues. The histidine-bound phosphate can be donated to suitable phosphate acceptors in a rapid reaction. This phosphotransfer reaction is highly nucleotide selective, as only ADP, but none of the other nucleoside-diphosphates tested, can be used as a phosphate acceptor.
Metabolism in trypanosomatids is compartmentalised with major pathways, notably glycolysis, present in peroxisome-like organelles called glycosomes. To date, little information is available about the transport of metabolites through the glycosomal membrane. Previously, three ATP-binding cassette (ABC) transporters, called GAT1-3 for Glycosomal ABC Transporters 1 to 3, have been identified in the glycosomal membrane of Trypanosoma brucei. Here we report that GAT1 and GAT3 are expressed both in bloodstream and procyclic form trypanosomes, whereas GAT2 is mainly or exclusively expressed in bloodstream-form cells. Protease protection experiments showed that the nucleotide-binding domain of GAT1 and GAT3 is exposed to the cytosol, indicating that these transporters mediate the ATP-dependent uptake of solutes from the cytosol into the glycosomal lumen. Depletion of GAT1 and GAT3 by RNA interference in procyclic cells grown in glucose-containing medium did not affect growth. Surprisingly, GAT1 depletion enhanced the expression of the very different GAT3 protein. Expression knockdown of GAT1, but not GAT3, in procyclic cells cultured in glucose-free medium was lethal. Depletion of GAT1 in glucose-grown procyclic cells caused a modification of the total cellular fatty-acid composition. No or only minor changes were observed in the levels of most fatty acids, including oleate (C18:1), nevertheless the linoleate (C18:2) abundance was significantly increased upon GAT1 silencing. Furthermore, glycosomes purified from procyclic wild-type cells incorporate oleoyl-CoA in a concentration- and ATP-dependent manner, whilst this incorporation was severely reduced in glycosomes from cells in which GAT1 levels had been decreased. Together, these results strongly suggest that GAT1 serves to transport primarily oleoyl-CoA, but possibly also other fatty acids, from the cytosol into the glycosomal lumen and that its depletion results in a cellular linoleate accumulation, probably due to the presence of an active oleate desaturase. The role of intraglycosomal oleoyl-CoA and its essentiality when the trypanosomes are grown in the absence of glucose, are discussed.
Mecanografiado Tesis (Lic. en Biología) -- Universidad de Los Andes, Facultad de Ciencias, Departamento de Biología, Mérida, 2006 Incluye bibliografía
Unlike other eukaryotic cells, trypanosomes possess a compartmentalized glycolytic pathway. The conversion of glucose into 3-phosphoglycerate takes place in specialized peroxisomes, called glycosomes. Further conversion of this intermediate into pyruvate occurs in the cytosol. Due to this compartmentation, many regulatory mechanisms operating in other cell types cannot work in trypanosomes. This is reflected by the insensitivity of the glycosomal enzymes to compounds that act as activity regulators in other cell types. Several speculations have been raised about the function of compartmentation of glycolysis in trypanosomes. We calculate that even in a noncompartmentalized trypanosome the flux through glycolysis should not be limited by diffusion. Therefore, the sequestration of glycolytic enzymes in an organelle may not serve to overcome a diffusion limitation. We also search the available data for a possible relation between compartmentation and the distribution of control of the glycolytic flux among the glycolytic enzymes. Under physiological conditions, the rate of glycolytic ATP production in the bloodstream form of the parasite is possibly controlled by the oxygen tension, but not by the glucose concentration. Within the framework of Metabolic Control Analysis, we discuss evidence that glucose transport, although it does not qualify as the sole rate-limiting step, does have a high flux control coefficient. This, however, does not distinguish trypanosomes from other eukaryotic cell types without glycosomes.
Over the last decade, our knowledge of the biochemistry and molecular biology of trypanosomes has expanded so much that the African trypanosome, Trypanosoma brucei, is now the equivalent of E. coli to the biochemical parasitologist. Trypanosomes are of interest to scientists not only because of their medical and veterinary importance, but also because of several unique features of their biochemistry and molecular biology. Two such features have been reviewed recently: the mitochondrial DNA network (the kinetoplast) and its role in the life cycle (Hajduk, 1978; Borst and Hoeijmakers, 1979; Barker, 1980; Englund et al., 1982), and the variant surface glycoprotein and its role in evading the immune response of the host (Englund et al., 1982; Turner, 1982).
The glycolytic enzyme glucosephosphate isomerase (PGI) is present in two different cell compartments of Leishmania mexicana promastigotes; more than 90% of the activity was detected in the cytosol, the remainder in glycosomes. This subcellular distribution contrasts with that in Trypanosoma brucei, in which the enzyme activity has been mainly located in the glycosomes. PGI was partially purified from L. mexicana cell extracts. Throughout the purification procedure only one single PGI activity could be detected. The partially purified protein had the same subunit molecular mass (65 kDa) as the previously characterized glycosomal protein of T. brucei. Both proteins were also very similar with respect to their kinetic and antigenic properties. Using the T. brucei glycosomal PGI gene as a hybridization probe, we cloned the corresponding gene of L. mexicana. Only a single PGI locus could be detected in the L. mexicana genome. Characterization of the cloned gene showed that it codes for a polypeptide of 604 amino acids, with a molecular mass of 67 113. The sequences of the Leishmania and Trypanosoma polypeptides are 69% identical. They differ in calculated net charge (−8 versus −2, respectively) and isoelectric point (6.65 versus 7.35). Our data strongly suggest that the PGI activity in the two cell compartments of L. mexicana and T. brucei is not attributable to different isoenzymes. We discuss the possible metabolic function of the highly different enzyme distribution in the two organisms, and the molecular mechanism that could be responsible for it.
We have studied the kinetics of the allosteric interactions of pyruvate kinase from Trypanosoma brucei. The kinetics for phosphoenolpyruvate depended strongly on the nature of the bivalent metal ions. Pyruvate kinase activated by Mg2+ had the highest catalytic activity, but also the highest S0.5 for phosphoenolpyruvate, while the opposite was true for pyruvate kinase activated by Mn2+. The reaction rates of Mg(2+)-pyruvate kinase and Mn(2+)-pyruvate kinase were clearly allosteric with respect to phosphoenolpyruvate, while the kinetics with Co(2+)-pyruvate kinase were hyperbolic. However, Co(2+)-pyruvate kinase was still sensitive to heterotropic activation. Trypanosomal pyruvate kinase is unique in that the best activator was fructose 2,6-bisphosphate. Ribulose 1,5-bisphosphate and 5-phosphorylribose 1-pyrophosphate were also strong heterotropic activators, which were much more effective than fructose 1,6-bisphosphate and glucose 1,6-bisphosphate. In the presence of the heterotropic activators, the sigmoidal kinetics with respect to phosphoenolpyruvate and the bivalent metal ions were modified as were the concentrations of phosphoenolpyruvate and the bivalent metal ions needed to attain the maximal activity. Maximal activities were not significantly changed with Mg2+ and Mn2+ as the activating metal ions. Moreover, with Co2+ and fructose 2,6-bisphosphate or ribulose 1,5-bisphosphate or 5-phosphorylribose 1-pyrophosphate, the maximal activity was significantly reduced. Ribulose 1,5-bisphosphate and 5-phosphorylribose 1-pyrophosphate resembled fructose 2,6-bisphosphate rather than fructose 1,6-bisphosphate and glucose 1,6-bisphosphate in their action in that the K0.5 values for the former 3 compounds increased when Mg2+ was replaced by Co2+, while the K0.5 for fructose 1,6-bisphosphate and glucose 1,6-bisphosphate increased.(ABSTRACT TRUNCATED AT 250 WORDS)
The mutual adjustment of glucose uptake and metabolism in the insect stage of the protozoan parasite Trypanosoma brucei was studied. T. brucei was preadapted in the chemostat to conditions in which either glucose or proline served as the major carbon and energy source. Cells were grown and adapted to either energy or non-energy limitation at a low dilution rate (0.5 day-1) or a high dilution rate (1 day-1). The cells were then used in short- to medium-term uptake experiments with D-[14C]glucose as a tracer. In time course experiments a steady state was reached after 15 min regardless of the preadaptation conditions. This steady-state level increased with increasing glucose availability during preadaptation. The rate of glucose uptake and the hexokinase activity were linearly correlated. In short-term 5- to 90-s) uptake experiments a high transport rate was measured with cultures grown in excess glucose, an intermediate rate was measured with proline-grown cultures, and a low rate was measured in organisms grown under glucose limitation. Glucose metabolism and proline metabolism did not affect each other during the 15-min incubations. Glucose uptake, as a function of the external glucose concentration, did not obey simple Michaelis-Menten kinetics but could be described by a two-step mechanism: (i) transport of glucose by facilitated diffusion and (ii) subsequent metabolism of glucose. The respective rates of the two steps were adjusted to each other. It is concluded that T. brucei is capable of adjusting the different metabolic processes in a way that gives maximum energy efficiency at the cost of short-term flexibility.
Trypanosoma brucei procyclic trypomastigotes were made permeable by using digitonin (0-70 micrograms/mg of protein). This procedure allowed exposure of coupled mitochondria to different substrates. Only succinate and glycerol phosphate (but not NADH-dependent substrates) were capable of stimulating oxygen consumption. Fluorescence studies on intact cells indicated that addition of succinate stimulates NAD(P)H oxidation, contrary to what happens in mammalian mitochondria. Addition of malonate, an inhibitor of succinate dehydrogenase, stimulated NAD(P)H reduction. Malonate also inhibited intact-cell respiration and motility, both of which were restored by further addition of succinate. Experiments carried out with isolated mitochondrial membranes showed that, although the electron transfer from succinate to cytochrome c was inhibitable by antimycin, NADH-cytochrome c reductase was antimycin-insensitive. We postulate that the NADH-ubiquinone segment of the respiratory chain is replaced by NADH-fumarate reductase, which reoxidizes the mitochondrial NADH and in turn generates succinate for the respiratory chain. This hypothesis is further supported by the inhibitory effect on cell growth and respiration of 3-methoxyphenylacetic acid, an inhibitor of the NADH-fumarate reductase of T. brucei.
CoQ links the sn-glycerol-3-phosphate dehydrogenase and oxidase components of the cyanide-insensitive, non-cytochrome-mediated respiratory system of bloodstream African trypanosomes. In this and other characteristics, their respiratory system is similar to the alternative oxidase of plants.
The parasites contain 206 ng of CoQ9 mg protein⁻¹ which co-sediments with respiratory activity. The redox state of this CoQ responds in a manner consistent with respiratory function: 60% being in the reduced form when substrate is available and the oxidase is blocked; 13% being in the reduced form when the oxidase is functioning and there is no substrate. The addition of CoQ to aceton-extracted cells stimulates salicylhydroxamic acid-sensitive respiration by 56%. After inhibition of respiration by digitonin-mediated dispersal of the electron transport components, liposomes restore 40% of respiratory activity while liposomes containing CoQ restore 66% of this activity. A less hydrophobic analogue, reduced decyl CoQ, serves as a direct substrate for the trypanosome oxidase supporting full salicylhydroxamic acid-sensitive respiration. After digitonin disruption of electron transport, the nonreduced form of this synthetic substrate can reestablish the chain by accepting electrons from dispersed sn-glycerol-3-phosphate dehydrogenase and transferring them to the dispersed oxidase.
Similarities between the alternative oxidase of plants and the oxidase of the trypanosome respiratory system include: mitochondrial location, lack of oxidative phosphorylation, linkage of a dehydrogenase and an oxidase by CoQ, lack of sensitivity to a range of mitochondrial inhibotors, and sensitivity to a spectrum of inhibitors which selectively block transfer of electrons from reduced CoQ to the terminal oxidase but do not block electron transfer to the cytochrome bc1 complex of the mammalian cytochrome chain.
Highly purified glycosomes were isolated from Trypanosoma brucei bloodstream forms and cultured procyclic trypomastigotes. A comparison of the specific activities of glycosomal enzymes revealed that glycosomes from insect stages had decreased levels of hexokinase, phosphoglucose isomerase, phospho-fructokinase, fructose-bisphosphate aldolase, glyceraldehyde-phosphate dehydrogenase and phosphoglycerate kinase, but contained increased levels of adenylate kinase, malate dehydrogenase and phosphoenolpyruvate carboxykinase. Glycosomes from bloodstream forms were almost totally devoid of the latter two activities. Comparison of the two types of glycosomes by sodium dodecylsulphate-polyacrylamide gel electrophoresis revealed that bloodstream form glycosomes contained 3 prominent polypeptides (64, 46 and 40 kDa) which were hardly detectable in insect stage glycosomes, whereas the latter contained 3 insect stage specific bands with molecular weight of 34 000, 61 000 and 77 000 and 4 additional bands with molecular weights between 94 000 and 110 000. Both types of glycosome contained the phospholipids phosphatidylcholine and phosphatidylethanolamine. Insect stage glycosomes contained in addition also phosphatidylinositol and some phosphatidylserine.
Procyclic culture forms of Trypanosoma brucei stock 427 have been screened for the presence of enzymes involved in glycolysis, mitochondrial energy metabolism and threonine degradation. The enzyme activities in the procyclics were compared with those of the blood stream forms. The specific activities of glycolytic enzymes represented 30-70% of the respective levels in the blood stream form, except for hexokinase which was 25-fold reduced. Cell fractionation showed that the enzymes involved in the early sequence of the glycolytic pathway, i.e. from hexokinase to phosphoglycerate kinase, and the enzymes NAD+-linked glycerol-3-phosphate dehydrogenase and glycerol kinase were all present in glycosomes equilibrating at a density of 1.23 g/cm3 in sucrose gradients. Malate dehydrogenase was 8-fold more active in procyclics than in bloodstream forms. This increase in activity was the result of the appearance of malate dehydrogenase in the glycosomes of the procyclics, in addition to mitochondrial and cell-sap activities which were present in both stages of the life cycle. Glycosomes contained part of the adenylate kinase activity, which was also associated with the mitochondrion. Succinate dehydrogenase and sn-glycerol-3-phosphate dehydrogenase, together with oligomycin-sensitive ATPase, were located in the mitochondrion which had a density in sucrose ranging from 1.16 to 1.18 g/cm3. This organelle also contained L-threonine 3-dehydrogenase and carnitine acetyltransferase, two enzymes involved in threonine catabolism. The latter two enzymes had activities which were, respectively, 15-and 13-fold higher in the procyclics than in the bloodstream form. Mitochondrial sn-glycerol-3-phosphate dehydrogenase was decreased 4-fold.
Alkyl-dihydroxyacetone phosphate synthase (E.C. 2.5.1.26), the key enzyme in ether phospholipid biosynthesis, was demonstrated to be present in Trypanosoma brucei. The distribution of alkyl-dihydroxyacetone phosphate synthase was found to be identical to that of dihydroxyacetone phosphate acyltransferase (E.C. 2.3.1.42), which has previously been shown to be exclusively associated with the glycosome fraction (Opperdoes, F.R. (1984) FEBS Lett. 169, 35-39). Studies with gradient purified glycosomes indicated that the formation of alkyl-dihydroxyacetone phosphate was completely dependent on the presence of acyl-dihydroxyacetone phosphate. The glycosomal alkyl-dihydroxyacetone phosphate synthase activity was characterized with respect to its pH optimum, Triton X-100 sensitivity and the dependency on the concentration of the substrates palmitoyl-dihydroxyacetone phosphate and hexadecanol. Using thin-layer chromatographic and alkaline hydrolysis procedures the reaction product was identified as alkyl-dihydroxyacetone phosphate. Alkyl-dihydroxyacetone phosphate synthase was resistant to proteolytic inactivation by trypsin in intact glycosomes but not in Triton X-100 disrupted glycosomes. It is concluded that T. brucei glycosomes contain the enzymes responsible for glycero-ether bond formation analogous to mammalian peroxisomes.
We investigated how NADH generated during peroxisomal beta-oxidation is reoxidized to NAD+ and how the end product of beta-oxidation, acetyl-CoA, is transported from peroxisomes to mitochondria in Saccharomyces cerevisiae. Disruption of the peroxisomal malate dehydrogenase 3 gene (MDH3) resulted in impaired beta-oxidation capacity as measured in intact cells, whereas beta-oxidation was perfectly normal in cell lysates. In addition, mdh3-disrupted cells were unable to grow on oleate whereas growth on other non-fermentable carbon sources was normal, suggesting that MDH3 is involved in the reoxidation of NADH generated during fatty acid beta-oxidation rather than functioning as part of the glyoxylate cycle. To study the transport of acetyl units from peroxisomes, we disrupted the peroxisomal citrate synthase gene (CIT2). The lack of phenotype of the cit2 mutant indicated the presence of an alternative pathway for transport of acetyl units, formed by the carnitine acetyltransferase protein (YCAT). Disruption of both the CIT2 and YCAT gene blocked the beta-oxidation in intact cells, but not in lysates. Our data strongly suggest that the peroxisomal membrane is impermeable to NAD(H) and acetyl-CoA in vivo, and predict the existence of metabolite carriers in the peroxisomal membrane to shuttle metabolites from peroxisomes to cytoplasm and vice versa.
Drug resistance is a significant impediment to the therapy of African sleeping sickness in humans. To evaluate molecular mechanisms that contribute to drug resistance in African trypanosomes, a clonal strain of Trypanosoma brucei gambiense, MPA10, was selected in medium containing mycophenolic acid (MPA), an inhibitor of IMP dehydrogenase (IMPDH) activity. IMPDH activity in MPA10 cells was approximately 6-fold higher than that of wild type parental cells, although the enzymes in both strains were equally sensitive to inhibition by MPA. To evaluate the mechanism of IMPDH overexpression in MPA10 cells, the gene encoding IMPDH (impdh) was isolated from a T.b. brucei library by cross-hybridization to the Leishmania donovani impdh. Sequence analysis indicated that the T. brucei IMPDH was 76% identical with the L. donovani IMPDH. The T. brucei impdh hybridized to a 2.7-kb transcript that was expressed at approximately 10-fold greater levels in the MPA10 cells, and this impdh overexpression could be ascribed to an approximately 10-fold amplification of the impdh copy number. Pulsed field gel electrophoresis revealed that the extra impdh copies in MPA10 cells were localized to an approximately 6.0-Mb chromosome that comigrated with the wild type chromosome encompassing impdh. The amplification of impdh could be ascribed to an increased copy number of this 6.0-Mb chromosome, and a predicted augmented DNA content in MPA10 cells was confirmed by flow cytometry. This is the first demonstration that DNA amplification can serve as a molecular mechanism by which T. brucei become resistant to cytotoxic drugs, and the amplification of the 6.0-Mb chromosome represents a novel mechanism of drug resistance in parasitic protozoa.
The kinetoplastid protozoa infect hosts ranging from invertebrates to plants and mammals, causing diseases of medical and economic importance. They are the earliest-branching organisms in eucaryotic evolution to have either mitochondria or peroxisome-like microbodies. Investigation of their protein trafficking enables us to identify characteristics that have been conserved throughout eucaryotic evolution and also reveals how far variations, or alternative mechanisms, are possible. Protein trafficking in kinetoplastids is in many respects similar to that in higher eucaryotes, including mammals and yeasts. Differences in signal sequence specificities exist, however, for all subcellular locations so far examined in detail--microbodies, mitochondria, and endoplasmic reticulum--with signals being more degenerate, or shorter, than those of their higher eucaryotic counterparts. Some components of the normal array of trafficking mechanisms may be missing in most (if not all) kinetoplastids: examples are clathrin-coated vesicles, recycling receptors, and mannose 6-phosphate-mediated lysosomal targeting. Other aspects and structures are unique to the kinetoplastids or are as yet unexplained. Some of these peculiarities may eventually prove to be weak points that can be used as targets for chemotherapy; others may turn out to be much more widespread than currently suspected.
Dihydrofolate reductase fusion proteins have been widely used to study conformational properties of polypeptides translocated across membranes. We have studied the import of dihydrofolate reductase fusion proteins into glycosomes and mitochondria of Trypanosoma brucei. As signal sequences we used the last 22 carboxy-terminal amino acids of glycosomal phosphoglycerate kinase for glycosomes, and the cleavable presequences of yeast cytochrome b2 or cytochrome oxidase subunit IV for mitochondria. Upon addition of aminopterin, a folate analogue that stabilizes the dihydrofolate reductase moiety, import of the fusion protein targeted to glycosomes was not inhibited, although the results of protease protection assays showed that the fusion protein could bind the drug. Under the same conditions, import of a DHFR fusion protein targeted to mitochondria was inhibited by aminopterin. When DHFR fusion proteins targeted simultaneously to both glycosomes and mitochondria were expressed, import into mitochondria was inhibited by aminopterin, whereas uptake of the same proteins into glycosomes was either unaffected or slightly increased. These findings suggest that the glycosomes possess either a strong unfolding activity or an unusually large or flexible translocation channel.
The glycosome, a microbody organelle found only in kinetoplastid protozoa, compartmentalizes the first six enzymes of glycolysis. In order to better understand the regulation and targeting of glycolytic enzymes in trypanosomes, we have cloned and analyzed the three genes of the phosphoglycerate kinase (PGK) complex of Trypanosoma (Nannomonas) congolense . The organization of the genes within the complex is similar to that of Trypanosoma brucei brucei . The nucleotide and amino-acid sequences, including those of the novel high-molecular-weight 56PGK, show substantial cross-species similarity. However, the two downstream genes, c1PGK and c2PGK , encode identical isozymes in T. congolense , while they encode distinct glycosomal and cytoplasmic isozymes in T. brucei . Western analysis also indicated that there are only two isozymes in T. congolense and that these are constitutively expressed. Differential digitonin solubilization of the trypanosomes indicated that 56PGK is primarily localized to the glycosome, as expected, and that c1 c2PGK is cytoplasmic. Northern analysis demonstrates that while 56PGK is constitutively expressed, c1PGK and c2PGK mRNAs are differentially expressed in the T. congolense developmental stages. This work demonstrates that T. congolense has only one PGK isozyme, 56PGK, that is predominantly localized in glycosomes.
Bloodstream forms of Trypanosoma brucei were found to maintain a significant membrane potential across their mitochondrial inner membrane (ΔΨm) in addition to a plasma membrane potential (ΔΨp). Significantly, the ΔΨm 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 150mV. When the value for the ΔΨm 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.
d ‐Glucose and 2‐deoxyglucose enter the ‘long‐slender’ bloodstream from of Trypanosoma brucei only by means of a carrier‐mediated process; no free diffusion can be observed. Permeation is not energy‐dependent. The uptake is driven by the downhill concentration gradient of free substrate. The latter is maintained by the continual removal of sugar, due to the extremely high activities of the glycolytic enzymes. The permeation process is the rate‐limiting step of glucose consumption, because permeation proceeds at a rate slower than metabolism.
The inhibition of sugar uptake by glycerol was tested. Interactions at the carrier site can be ruled out since glucose and its 2‐deoxy analog exhibit different inhibition kinetics.
When the infected mammalian host of Trypanosoma brucei brucei is injected with a solution of the iron chelator salicyl hydroxamic acid and glycerol, the aerobic and anaerobic glucose catabolism of the parasite is blocked and the parasite is rapidly destroyed.
The consumption of glucose by trypanosomatid protozoa such as Trypanosoma brucei, Trypanosoma cruzi, Leishmania spp., and Crithidia spp. is characterized by the excretion of reduced products such as succinate, pyruvate, ethanol, L-alanine, or lactate (depending on the species) not only in anaerobiosis, but also under aerobic conditions. The "aerobic fermentation" of glucose is accompanied by a complete lack, or even a reversal, of the Pasteur effect. This peculiar catabolism is mediated by a so-far unique compartmentation of the glycolytic enzymes, most of which are placed in an organelle called the glycosome; by an almost complete lack of inhibitory controls at the level of hexokinase and phosphofructokinase; and by a central role of CO2 fixation through the reaction catalyzed by phosphoenolpyruvate carboxykinase. The production of fermentative products seems to be due to a relative inefficiency of the respiratory chain, which lacks NADH dehydrogenase and the first phosphorylation site and preferentially uses succinate as substrate.
Intermediate and short stumpy bloodstream forms of Trypanosoma brucei brucei are transitional stages in the differentiation of mammal-infective long slender bloodstream forms into the procyclic forms found in the midgut of the tsetse vector. Although the mitochondria of the proliferative long slender forms do not accumulate rhodamine 123, the mitochondria of the transitional forms attain this ability thus revealing the development of an electromotive force (EMF) across the inner mitochondrial membrane. The EMF is inhibited by 2,4-dinitrophenol, rotenone and salicylhydroxamic acid but not by antimycin A or cyanide. Consequently, NADH dehydrogenase, site I of oxidative phosphorylation, is the source of the EMF and the plant-like trypanosome alternative oxidase (TAO) supports the electron flow serving as the terminal oxidase of the chain. Although the TAO is present in the long slender forms as well, it serves only as the terminal oxidase for electrons from glycerol-3-phosphate dehydrogenase. The data presented here, combined with older data, lead to the conclusion that the mitochondria of transitional intermediate and short stumpy forms likely produce ATP. This putative production is either by F1F0 ATPase driven by the complex I proton pump or by mitochondrial substrate level phosphorylation, or most likely by both. These conclusions contrast with the previously held dogma that all bloodstream form mitochondria are incapable of ATP production.
The subcellular localization of NAD- and NADP-linked glutamate dehydrogenases (GDH-NAD and GDH-NADP), alanine aminotransferase (ALAT) and aspartate aminotransferase (ASAT) in epimastigotes of Trypanosoma cruzi was studied by digitonin extraction from whole cells, subcellular fractionation by differential centrifugation and isopycnic ultracentrifugation. All enzymes presented both a cytosolic and a mitochondrial form; in addition, GDH-NADP seems to have a third, still undefined, localization. The results are compatible with the existence of two pathways for the production of L-alanine linked to the reoxidation of glycolytic NADH, one operative in the mitochondrion and the other in the cytosol, and perhaps responsible for the existence of the two alanine pools detected by 13C-nuclear magnetic resonance (B. Frydman et al., Eur. J. Biochem. 192 (1990) 363-368).
We report the differential expression of the oligomycin-sensitive mitochondrial ATPase in pleomorphic bloodstream forms of Trypanosoma brucei brucei as observed with enzymatic assays and electron microscope histochemistry. As the cells differentiate from long slender to short stumpy forms, total specific activity of the mitochondrial ATPase in a crude mitochondrial fraction doubles and the oligomycin-sensitive specific activity increases 5-fold. Upon in vitro differentiation to procyclic forms, there is a further doubling of total specific activity and a further tripling of oligomycin-sensitive specific activity. The oligomycin-insensitive ATPase activity remained essentially constant throughout differentiation. We have attempted to characterize this oligomycin-insensitive activity utilizing inhibitors of several other ATPases.
The phosphoglycerate kinase (PGK) gene complex of Trypanosoma brucei contains three tandemly linked related genes. One gene encodes a cytoplasmic PGK, while another encodes a PGK isozyme localized to glycosomal microbodies. In this communication, we report that the third gene in this complex encodes a 56-kDa molecule which is also localized to the glycosomal core. DNA sequence analysis indicates that this gene contains multiple substitutions and a large insertion in the amino domain, but that it is very similar to the other PGK isozymes in the carboxy domain. The C-terminal tripeptide is identical to that of the cytoplasmic isozyme, and only one conservative change occurs in the last 25 amino acids. The encoded protein, p56, thus contrasts with the many peroxisomal microbody proteins in which the C-terminal tripeptide contains sufficient information for targeting to peroxisomes. Multiple mechanisms may exist for targeting proteins to the protein cores of microbody organelles. Comparisons of the DNA sequences of several alleles suggest that homologous recombination plays a role in the generation of allelic diversity.
A rapid switch from a fermentative to a primarily oxidative type of glucose utilization was observed during in vitro differentiation of Trypanosoma brucei STIB348 and EATRO1244 bloodstream to procyclic trypomastigotes. In accordance with previously published reports bloodstream populations produced pyruvate as the major end product of glucose catabolism, together with very small amounts of CO2, succinate and glycerol. During differentiation pyruvate excretion decreased within 48 h to the low levels produced by 28-day procyclic stages. Concomitant with the decline in pyruvate formation, acetate appeared as a new product and the rates of respiratory CO2 increased considerably. The amount of carbon released with these compounds could account for nearly all of the glucose carbon consumed. Rates of glucose utilization and formation of acetate and CO2 in cells differentiated for 48 h were essentially the same as those found in 28-day procyclics. Succinate and glycerol excretion remained low during the entire transformation process, and no significant difference in the pattern and quantities of end products were found between the two trypanosome strains. During trypanosome differentiation the changes in metabolism were associated with marked alterations in enzyme activity levels. Activities of the tricarboxylic acid (TCA) cycle enzymes citrate synthase, isocitrate dehydrogenase (NAD+), succinate dehydrogenase and fumarase were not detectable in bloodstream trypomastigotes but appeared upon differentiation for 24 h. An exception was citrate synthase whose activity was not demonstrable until 48 h postinoculation into culture. After 48 h the majority of the TCA cycle enzyme activities continued to increase steadily until day 28. Pyruvate kinase activity decreased in differentiating cells after 48 h to about 25% of the level found in bloodstream trypomastigotes.(ABSTRACT TRUNCATED AT 250 WORDS)
The specific activities of each of the enzymes of the classical pentose phosphate pathway have been determined in both cultured procyclic and bloodstream forms of Trypanosoma brucei. Both forms contained glucose-6-phosphate dehydrogenase (EC 1.1.1.49), 6-phosphogluconolactonase (EC 3.1.1.31), 6-phosphogluconate dehydrogenase (EC 1.1.1.44), ribose-5-phosphate isomerase (EC 5.3.1.6) and transaldolase (EC 2.2.1.2). However, ribulose-5-phosphate 3'-epimerase (EC 5.1.3.1) and transketolase (EC 2.2.1.1) activities were detectable only in procyclic forms. These results clearly demonstrate that both forms of T. brucei can metabolize glucose via the oxidative segment of the classical pentose phosphate pathway in order to produce D-ribose-5-phosphate for the synthesis of nucleic acids and reduced NADP for other synthetic reactions. However, only procyclic forms are capable of using the non-oxidative segment of the classical pentose phosphate pathway to cycle carbon between pentose and hexose phosphates in order to produce D-glyceraldehyde 3-phosphate as a net product of the pathway. Both forms lack the key gluconeogenic enzyme, fructose-bisphosphatase (EC 3.1.3.11). Consequently, neither form should be able to engage in gluconeogenesis nor should procyclic forms be able to return any of the glyceraldehyde 3-phosphate produced in the pentose phosphate pathway to glucose 6-phosphate. This last specific metabolic arrangement and the restriction of all but the terminal steps of glycolysis to the glycosome may be the observations required to explain the presence of distinct cytosolic and glycosomal isoenzymes of glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase. These same observations also may provide the basis for explaining the presence of cytosolic hexokinase and phosphoglucose isomerase without the presence of any cytosolic phosphofructokinase activity. The key enzymes of the Entner-Doudoroff pathway, 6-phosphogluconate dehydratase (EC 4.2.1.12) and 2-keto-3-deoxy-6-phosphogluconate aldolase (EC 4.1.2.14) were not detected in either procyclic or bloodstream forms of T. brucei.
The expression of procyclic acidic repetitive protein (PARP) by Trypanosoma brucei is strongly induced during the transition
of bloodstream form to cultured procyclic trypomastigotes in vitro. The membrane-associated protein is distinguished by a
central domain consisting of tandemly repeated glutamate-proline dipeptides. The trypanosome genome contains eight PARP genes,
at least four of which are expressed. A minimum of four distinct PARP mRNA species comprises two classes of PARP mRNA, based
upon divergent 3' untranslated region sequences, and these mRNAs encode polypeptides that exhibited an inverse relation between
molecular weight and isoelectric point. Comparative analysis of PARP gene structure indicated that these polypeptides differ
by variation in size of the dipeptide repeat domain. Comparison of PARP genes and polypeptides of three independent T. brucei
isolates suggested that PARP is not a homogeneous species but instead represents a family of polymorphic proteins.
SYNOPSIS
Exponentially dividing culture forms of Trypanosoma brucei did not utilize glucose provided in the culture medium. The inclusion of 2‐deoxyglucose in the medium had no effect on the growth of the trypanosomes. Glucose could be replaced by proline in the liquid phase of biphasic medium without affecting the doubling time of the organisms. Proline added to the culture medium in this way disappeared during the log phase of growth. Glucose in the culture medium was used by the trypanosomes only when the stationary growth phase had been reached. Lipid accumulated in stationary phase trypanosomes grown in glucose‐containing medium, but there was no lipid accumulation in log phase organisms or in those which had been grown in proline‐containing medium. Bloodstream trypanosomes transferred to liquid medium rapidly utilized glucose over the first 12 hr of culture, and this was accompanied by an accumulation of free pyruvate in the medium. The rate of glucose utilization fell off over the next 36 hr; this was accompanied by a lowering of free pyruvate in the medium and a rise in the proline oxidase activity of the trypanosomes. The possible biologic significance of proline to trypanosomes developing in the midgut of the tsetse vector is discussed.
In cell-fractionation experiments most of the glycolytic enzymes in bloodstream forms of Trypanosoma brucei are recovered in a microbody, called the glycosome [Opperdoes, F. R. and Borst, P. (1977) FEBS Lett. 80, 360–364]. To see whether this compartmentation of glycolytic enzymes is accompanied by compartmentation of metabolites we have pulse-labelled intact T. brucei with [U-¹⁴C]glucose and followed the incorporation of radioactivity into glycolytic intermediates separated by anion-exchange chromatography. The kinetics of incorporation provide direct evidence for the existence of two pools of glycolytic intermediates. One pool is completely labelled within 15 s. and represents 20–30% of total cellular metabolites. Radioactively labelled pyruvate is already produced after 15 s. Since this pool is directly involved in the glycolytic flux, we conclude that it is present in the glycosome. The second pool which represents 70–80 % of the total appears not to be directly involved in glycolysis. Its content equilibrates relatively slowly with the glycosomal pool. It probably represents the cytosol.
The kinetoplastid protozoa infect hosts ranging from invertebrates to plants and mammals, causing diseases of medical and economic importance. They are the earliest-branching organisms in eucaryotic evolution to have either mitochondria or peroxisome-like microbodies. Investigation of their protein trafficking enables us to identify characteristics that have been conserved throughout eucaryotic evolution and also reveals how far variations, or alternative mechanisms, are possible. Protein trafficking in kinetoplastids is in many respects similar to that in higher eucaryotes, including mammals and yeasts. Differences in signal sequence specificities exist, however, for all subcellular locations so far examined in detail--microbodies, mitochondria, and endoplasmic reticulum--with signals being more degenerate, or shorter, than those of their higher eucaryotic counterparts. Some components of the normal array of trafficking mechanisms may be missing in most (if not all) kinetoplastids: examples are clathrin-coated vesicles, recycling receptors, and mannose 6-phosphate-mediated lysosomal targeting. Other aspects and structures are unique to the kinetoplastids or are as yet unexplained. Some of these peculiarities may eventually prove to be weak points that can be used as targets for chemotherapy; others may turn out to be much more widespread than currently suspected.
An inducible expression system was developed for the protozoan parasite Trypanosoma brucei. Transgenic trypanosomes expressing the tetracycline repressor of Escherichia coli exhibited inducer (tetracycline)-dependent expression of chromosomally integrated reporter genes under the control of a procyclic acidic repetitive protein (PARP) promoter bearing a tet operator. Reporter expression could be controlled over a range of four orders of magnitude in response to tetracycline concentration, a degree of regulation that exceeds those exhibited by other eukaryotic repression-based systems. The tet repressor-controlled PARP promoter should be a valuable tool for the study of trypanosome biochemistry, pathogenicity, and cell and molecular biology.
Glycosomes are microbodies found in protozoa belonging to the order Kinetoplastida. These highly specialized organelles compartmentalize most of the glycolytic enzymes normally located in the cytosol of other eukaryotic cells. The recent success in expressing foreign proteins in Trypanosoma brucei has permitted a detailed analysis of glycosomal protein targeting signals in these organisms. These studies have revealed that the previously identified C-terminal tripeptide peroxisomal targeting signal also functions in the import of proteins into the glycosomes of T. brucei. However, the glycosomal and peroxisomal targeting signals differ in a few important ways. The C-terminal tripeptide sequence requirements for glycosomal protein targeting are comparatively relaxed. Of the three C-terminal amino acids, the first can be any small, neutral amino acid; the second should be capable of forming hydrogen bondings, whereas the third is a hydrophobic amino acid. This degenerate tripeptide sequence differs significantly from the more stringent requirements observed for the import of proteins into mammalian peroxisomes and thus represents an opportunity for designing peptide analogues that specifically block the glycosomal protein import for a possible antitrypanosomal chemotherapy. A recently described N-terminal signal that targets thiolase to the mammalian peroxisomes does not appear to function in import into the glycosomes. However, a novel internal targeting signal has tentatively been identified in at least one of the glycosomal proteins that can target a reporter protein to the glycosomes of T. brucei. Glycosome-deficient mutants have been isolated recently, which will aid in the identification of genes involved in the biogenesis of the glycosome.
The glycosomes of trypanosomes are related to eukaryotic peroxisomes. For many glycosomal and peroxisomal proteins, a C-terminal SKL-like tripeptide known as PTS-1 serves as the targeting signal. For peroxisomes, a second N-terminal signal (PTS-2) was demonstrated on rat 3-ketoacyl-CoA thiolase. Several glycosomal proteins do not bear a PTS-1. One such protein, fructose bisphosphate aldolase, has a PTS-2 homology at its N-terminus. To find out whether the PTS-2 pathway exists in trypanosomes, we expressed chloramphenicol acetyltransferase fusion proteins bearing N-terminal segments of either rat thiolase or trypanosome aldolase. The mammalian PTS-2 clearly mediated glycosomal import. The aldolase N-terminus mediated import with variable efficiency depending on the length of the appended sequence. These results provide evidence for the existence of the PTS-2 pathway in trypanosomes.
The enzyme dihydrolipoamide dehydrogenase has been discovered and characterised in four salivarian trypanosomes of the subgenus trypanozoon: Trypanosoma brucei brucei, T. b. gambiense, T. b. rhodesiense, and Trypanosoma evansi. The three T. brucei species, which have insect procyclic forms biochemically distinct from their mammalian bloodstream forms, express dihydrolipoamide dehydrogenase in both cell types, but have higher levels in the procyclic forms. Determination of Michaelis constants for the enzyme from each of the three T. brucei species did not reveal any significant kinetic differences between the bloodstream and procyclic enzymes. On Western blots, antibodies raised against dihydrolipoamide dehydrogenase from the stereorarian trypanosome, Trypanosoma cruzi, cross-react strongly with the dihydrolipoamide dehydrogenase from all three T. brucei species; by this method, the relative molecular masses of their dihydrolipoamide dehydrogenases are indistinguishable. Dihydrolipoamide dehydrogenase was purified from both the bloodstream and the procyclic forms of T. b. brucei, and the N-terminal have been sequenced. These sequences are identical to the derived protein sequence of the cloned gene (Else et al., Eur. J. Biochem. 212 (1993) 423-429), but have a nine amino acid N-terminal truncation, giving an N-terminus equivalent to that of T. cruzi dihydrolipoamide dehydrogenase. The T. b. brucei dihydrolipoamide dehydrogenase gene has been expressed in Escherichia coli and the resultant protein purified; its N-terminus is processed in a similar fashion to that in the trypanosome, but with reduced specificity.
NADP-malic enzyme II, one of two isoenzymes of NADP-malic enzyme (EC 1.1.1.40) in Trypanosoma cruzi epimastigotes, presents hysteretic behavior that results in a kinetic lag in the reaction progress curve. The lag is affected by the malate, aspartate and oxaloacetate concentrations in the assay mixture. This dependence suggests that hysteresis is due to an association-dissociation process influenced by the binding of these ligands to the enzyme. The enzyme was separated from NADP-malic enzyme I and purified 43-fold from a cell homogenate by a procedure involving column chromatography on DEAE-Sephacel and Cibacron-blue Sepharose. The molecular mass of the highly purified enzyme was determined as 126 kDa.
The structure and regulation of the Trypanosoma brucei mitochondrial ATP synthase is reviewed. This enzyme complex which catalyzes the synthesis and hydrolysis of ATP within the mitochondrion is a multisubunit complex which is regulated in several ways. Several lines of evidence have shown that the ATP synthase is regulated through the life cycle of Trypanosoma brucei. The enzyme complex is present at maximal levels in the procyclic form where mitochondrial activity is the highest and cytochromes and Kreb's cycle components are present. The levels of the ATP synthase are decreased in the bloodstream forms where the levels of the mitochondrial cytochromes are absent or substantially decreased. In recent preliminary work we have shown the presence of an ATP synthase inhibitor peptide which may indicate an additional level of complexity to the regulation.
An electrochemical proton gradient exists across the plasma membrane and the mitochondrial membrane of the bloodstream form of Trypanosoma brucei. The membrane potential across the plasma membrane and the regulation of the internal pH depend on the temperature. Leishmania donovani regulates its internal pH and maintains a constant electrochemical proton gradient across its plasma membrane under all conditions examined. The mitochondrion of the T. brucei bloodstream form is energized, even though the reactions taking place in it do not result in net ATP synthesis and the Kreb's cycle and the respiratory chain are absent. Glucose is transported across the plasma membrane of T. brucei by a facilitated diffusion carrier, that can transport a wider range of substrates than its mammalian counterparts. Pyruvate exits the cell via a facilitated diffusion transporter as well. Conflicting evidence exists for the mechanism of glucose transport in L. donovani; biochemical evidence suggests proton/glucose symport, while facilitated diffusion is indicated by physiological data.
Epimastigotes of Trypanosoma cruzi, the causative agent of Chagas disease, catabolize proteins and amino acids with production of MH3, and glucose with production of reduced catabolites, chiefly succinate and L-alanine, even under aerobic conditions. This "aerobic fermentation of glucose" is probably due to both the presence of low levels of some cytochromes, causing a relative inefficiency of the respiratory chain for NADH, reoxidation during active glucose catabolism, and the lack of NADH dehydrogenase and phosphorylation site I, resulting in the entry of reduction equivalents into the chain mostly as succinate. Phosphoenol pyruvate carboxykinase and pyruvate kinase may play an essential role in diverting glucose carbon to succinate or L-alanine, and L-malate seems to be the major metabolite for the transport of glucose carbon and reduction equivalents between glycosome and mitochondrion. The parasite contains proteinase and peptidase activities. The major lysosomal cysteine proteinase, cruzipain, has been characterized in considerable detail, and might be involved in the host/parasite relationship, in addition to its obvious role in parasite nutrition. Among the enzymes of amino acid catabolism, two glutamate dehydrogenases (one NADP- and the other NAD-linked), alanine aminotransferase, and the major enzymes of aromatic amino acid catabolism (tyrosine aminotransferase and aromatic alpha-hydroxy acid dehydrogenase), have been characterized and proposed to be involved in the reoxidation of glycolytic NADH.
The pathway of NADH oxidation in the procyclic Trypanosoma brucei brucei was investigated in a crude mitochondrial membrane fraction and in whole cells permeabilized with digitonin. NADH:cytochrome c reductase activity was 75% inhibited by concentrations of antimycin that inhibited 95% succinate:cytochrome c reductase activity suggesting that the major pathway for NADH oxidation in the mitochondria involved the cytochrome bc1 complex of the electron transfer chain. Both NADH:cytochrome c and NADH:ubiquinone reductase activities were inhibited 80-90% by rotenone indicating the presence of a complex I-like NADH dehydrogenase in the mitochondrion of trypanosomes. In whole cells permeabilized with low concentrations of digitonin, the oxidation of malate, proline and glucose (in the presence of salicylhydroxamic acid, the inhibitor of the alternate oxidase) was inhibited 30-50% by rotenone. The presence of an alternative pathway for NADH oxidation involving fumarate reductase was indicated by the observation that malonate, the specific inhibitor of succinate dehydrogenase, inhibited 30-35% the rate of oxygen uptake with malate and glucose as substrates in the digitonin-permeabilized cells. We conclude that in the mitochondrion of the procyclic form of T. brucei, NADH is preferentially oxidized by a rotenone-sensitive NADH:ubiquinone oxidoreductase; however, NADH can also be oxidized to some extent by the enzyme fumarate reductase present in the mitochondrion of T. brucei.
There are 3 loci in the phosphoglycerate kinase (PGK) gene complex of Trypanosoma brucei. The PGK-A gene product, which we term 56PGK, is targeted to glycosomal microbodies and is highly homologous to the parasite's 2 known PGKs (one cytoplasmic and one glycosomal). However, 56PGK contains an 80 amino acid insertion as well as numerous substitutions compared to the other PGKs. The complementation and kinetic analyses described here demonstrate that 56PGK is an authentic phosphoglycerate kinase--the largest yet described. When expressed in Escherichia coli, 56PGK complements the pgk- phenotype. 56PGK was expressed as a fusion protein and purified to near homogeneity. The Michaelis constants are similar to those of other PGKs, being 0.12 and 2.4 mM for Mg-ATP and 3-phosphoglycerate, respectively. As with other T. brucei PGKs, ATP but not GTP or ITP can serve as a phosphate donor during catalysis. No evidence was obtained for phosphate transfer to atypical substrates. 56PGK shows sulfate inhibition at all concentrations tested, rather than the sulfate activation observed with yeast PGK.
A Trypanosoma brucei gene has been identified that encodes a protein predicted to be a component of the trypanosome homologue of mitochondrial NADH:ubiquinone oxidoreductase (complex I). High homology was found to a 20-kDa component of the iron-sulfur protein fraction of bovine mitochondrial NADH:ubiquinone oxidoreductase and the products of the ndhK locus of Paramecium tetraurelia mitochondria and the NQO6 locus of Paracoccus denitrificans. The homology extends to several other proteins predicted to function as part of electron transport systems, including the psbG/ndhK gene products of chloroplast and cyanobacterial genomes which are thought to be subunits of a NADH:plastoquinone oxidoreductase involved in chlororespiration. The T. brucei ndhK counterpart is nuclearly encoded. An extended amino terminus of the T. brucei ndhK with structural similarity to mitochondrial presequences indicates that its transfer into mitochondria is likely. Stumpy and slender bloodforms and procyclic forms all possess similar levels of ndhK transcripts despite previous reports of stage-regulated expression of complex I-like activity.
The gene encoding the hypoxanthine-guanine phosphoribosyltransferase (HGPRT) enzyme from Leishmania donovani has been cloned and sequenced. The hgprt open reading frame encoded a polypeptide of 211 amino acids that exhibited 3 regions of significant homology with other eukaryotic HGPRTs and a C-terminal tripeptide compatible with a glycosomal targeting signal. Northern blot analysis of L. donovani RNA revealed two hgprt transcripts, a 1.9-kb mRNA and a 1.7-kb transcript. The expression of the 1.7-kb hgprt mRNA and the activity of HGPRT enzyme were both augmented approx. 5-fold in parasites incubated in the absence of purines. Southern blots of genomic DNA indicated only a single hgprt locus within the L. donovani genome. Overexpression of L. donovani hgprt in E. coli complemented genetic deficiencies in hypoxanthine and guanine phosphoribosylating activities and yielded abundant quantities of enzymatically active HGPRT. The recombinant HGPRT was purified to homogeneity and recognized hypoxanthine, guanine and allopurinol, but not adenine or xanthine, as substrates. The hgprt clone and pure HGPRT protein provide essential reagents for validating HGPRT as a therapeutic target for the treatment of leishmaniasis and other diseases of parasitic origin.
The import of proteins into the glycosome of T. brucei has been studied as a potential target for antitrypanosomal chemotherapy. We have previously reported on the C-terminal tripeptides, such as SKL and its analogs, which function as targeting signals in the import of proteins into glycosomes. Recently, we tested the herpes simplex virus thymidine kinase gene (tk) as both a potential positive and a negative selectable marker in T. brucei for isolating glycosome-deficient T. brucei mutants in the procyclic form and complementation studies to identify genes involved in glycosomal biogenesis. The transforming vectors that we have constructed contained the hygromycin phosphotransferase gene (hyg) coupled either to the tk gene (ptk) or to the gene encoding the thymidine kinase fused with the glycosomal targeting signal (ptk-SKL) at the C-terminus. Individual constructs were introduced into the T. brucei 427 procyclic cells by electroporation, and the transformants were selected under hygromycin B (50 micrograms/ml) and cloned. The thymidine kinase activity in the crude extracts of transformants was determined. Differential digitonin treatment of the transformants indicated that the tk-SKL protein was apparently localized to the glycosomal fraction, as expected, whereas the tk protein was found in the soluble fraction. [methyl-3H]Thymidine was incorporated into the nucleic acids of the transformant 427/ptk to a level twice as high as that incorporated into 427/ptk-SKL and the wild type. In the presence of 100 microM ganciclovir, the growth of 427/ptk was totally inhibited, whereas growth of 427/ptk-SKL and the wild type was unaffected. When 150 microM trimethoprim was added to the culture medium, growth of 427/ptk-SKL and the wild type was completely arrested while that of 427/ptk remained normal. We have thus established the methodology for both positive and negative selection of potential glycosome-deficient mutants of T. brucei 427/ptk-SKL.
Inhibition analysis of respiration of Leishmania donovani promastigotes in resting, starved and permeabilized cells in the presence of classical electron transfer complex inhibitors such as rotenone, thenoyltrifluoroacetone and antimycin demonstrated the absence of complex I component of the respiratory chain in this organism. Cyanide failed to completely block the oxygen uptake (residual 25-30%) even at high concentrations. The alternative oxidase inhibitor for Trypanosoma brucei, salicylhydroxamic acid (SHAM) had no effect on respiration while the cytochrome o inhibitor orthohydroxydiphenyl (OHD) could block cyanide-insensitive respiration at low concentrations. Succinate-dependent O2 uptake in permeabilized cells follows the classical pathway. Oxidation of NADH by a membrane-rich fraction produced H2O2 as the end product and was insensitive to respiratory chain inhibitors. The presence of NADH-fumarate reductase was demonstrated in membrane-rich fraction and fumarate could reduce H2O2 production from NADH indicating fumarate to be an endogenous substrate for accepting electrons from NADH. A differential route for NADH oxidation was further confirmed by NADH cytochrome c reductase insensitivity to antimycin. A tentative scheme for electron transfer pathway in this organism is proposed in which a reversal of Krebs cycle enzymes occur producing succinate that can be excreted or oxidized depending upon the energy demands of the cell. Inhibition studies also suggest bifurcations of the respiratory chain that can be of minor importance for the organism.
~ (h'twnberg, I. Sharma, I'.R. and l)e~hu~,e,, l. (1~7~) D-glu-cose transport in 'i'rypanosoma brucei. Ettr. 1. BioHtcm. 8~J, -I o I -,.Io9 3~J ¢ aliens, M. and Opperdoes, I:.R. (I 9,~2) Some kinetic properties of pyruvate kinase from Trypanosoma brucei
- I~i~p
I~i~p,'m'l N. Bimm'ml,r. 27, 513-525 3,~ (h'twnberg, I. Sharma, I'.R. and l)e~hu~,e,,, l. (1~7~) D-glu-cose transport in 'i'rypanosoma brucei. Ettr. 1. BioHtcm. 8~J, -I o I -,.Io9 3~J ¢ aliens, M. and Opperdoes, I:.R. (I 9,~2) Some kinetic properties of pyruvate kinase from Trypanosoma brucei. Mol. Biochem. I'arasitol. 5(I, 235-244
Alkyl-dihydroxyacetone phosphate synthase in glyco-somes of Trypanosoma brttcei. Biochim. Bioph!/s. Acta 1257, 167-173 3¢~ Van den Bosch, I I. ctal
- A W Zomer
- F R Opperdoes
- Van
- Bosch
Zomer, A.W., Opperdoes, F.R. and van den Bosch, tt. (19951 Alkyl-dihydroxyacetone phosphate synthase in glyco-somes of Trypanosoma brttcei. Biochim. Bioph!/s. Acta 1257, 167-173 3¢~ Van den Bosch, I I. ctal. (19921 Biochemistry of peroxisomes.,4 ram. R,':'. Bi~ ~c/a'm. ~ 1, 157-197
D-glucose transport in Trypanosoma brucei
- Gruenberg