Active transport of 2-deoxy-d-glucose in Trypanosoma brucei procyclic forms

Parsons, Marilyn; Nielsen, Barbara

doi:10.1016/0166-6851(90)90162-f

Cited by 36 publications

(31 citation statements)

References 21 publications

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“…Kinetic Analysis of Glucose Uptake-Glucose transport was assayed by a rapid oil-stop method (6). Cells were washed once with PBS and resuspended in PBS to a density of 5 ϫ 10 7 cells/ml, and 100 l were layered over 200 l of a mixture of dibutylphthalate and light mineral oil (95:5 v/v) in a 1.5-ml Microfuge tube.…”

Section: Methodsmentioning

confidence: 99%

The Adenosine Analog Tubercidin Inhibits Glycolysis in Trypanosoma brucei as Revealed by an RNA Interference Library

Drew

Morris

Wang

et al. 2003

Journal of Biological Chemistry

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We used an RNA interference (RNAi) library in a forward genetic selection to study the mechanism of toxicity of tubercidin (7-deazaadenosine) to procyclic Trypanosoma brucei. Following transfection of cells with an RNAibased genomic library, we used 5 M tubercidin to select a drug-resistant cell line. Surprisingly, we found in these resistant cells that the hexose transporters had been silenced. We subsequently found that silencing of hexokinase, a glycolytic enzyme, also yielded tubercidinresistant parasites. These observations suggested that glycolysis could be a target of tubercidin action and that RNAi silencing of glycolytic enzymes was gradual enough to allow the parasites to adapt to alternative sources of energy. Indeed, adaptation of procyclic trypanosomes to a glucose-independent metabolism by reduction of glucose in the culture medium caused tubercidin resistance. High pressure liquid chromatography analysis of glycolytic intermediates from parasites treated with tubercidin showed a dose-dependent increase in concentration of 1,3-bisphosphoglycerate, a substrate of phosphoglycerate kinase. Furthermore, tubercidin triphosphate inhibited recombinant T. brucei phosphoglycerate kinase activity in vitro with an IC 50 of 7.5 M. We conclude that 5 M tubercidin kills trypanosomes by targeting glycolysis, especially by inhibition of phosphoglycerate kinase.

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Section: Methodsmentioning

confidence: 99%

The Adenosine Analog Tubercidin Inhibits Glycolysis in Trypanosoma brucei as Revealed by an RNA Interference Library

Drew

Morris

Wang

et al. 2003

Journal of Biological Chemistry

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“…Procyclic culture forms, homologous to the insect midgut stage, can thrive in the absence of glucose, preferring amino acids (mainly proline) as an energy source. Nevertheless, procyclic forms do express a glucose transport system which differs from that found in the bloodstream forms (31,38).In a previous report, we described the cloning and analysis of a developmentally regulated T. brucei gene coding for a putative hexose transporter called THT (for trypanosome hexose transporter) (9). Here we report the characterization of a multigene family encoding two highly related proteins, THT1 and THT2.…”

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confidence: 99%

Differential regulation of two distinct families of glucose transporter genes in Trypanosoma brucei.

Bringaud¹,

Baltz²

1993

Mol. Cell. Biol.

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A tandemly arranged multigene family encoding putative hexose transporters in Trypanosoma brucei has been characterized. It is composed of two 80%Yo homologous groups of genes called THT1 (six copies) and THT2 (five copies). When Xenopus oocytes are microinjected with in vitro-transcribed RNA from a THT1 gene, they express a glucose transporter with properties similar to those of the trypanosome bloodstream-form protein(s). This THT1-encoded transport system for glucose differs from the human erythrocyte-type glucose transporter by its moderate sensitivity to cytochalasin B and its capacity to transport D-fructose. These properties suggest that the trypanosomal transporter may be a good target for antitrypanosomal drugs. mRNA analysis revealed that expression of these genes was life cycle stage dependent. Bloodstream forms express 40-fold more THT1 than THT2. In contrast, procyclic trypanosomes express no detectable THT1 but demonstrate glucosedependent expression of THT2.Glucose is the major source of energy for most protozoan parasites. The uptake of this sugar across the plasma membrane is a process of fundamental importance. The wellcharacterized glucose transport systems of members from the genera Trypanosoma (14,16,18,36,38), Leishmania (46,47), and Plasmodium (37) are closely related, as judged by their substrate specificity and drug sensitivity. They act as either facilitative transporters (18,38) (14,16,36,38). Procyclic culture forms, homologous to the insect midgut stage, can thrive in the absence of glucose, preferring amino acids (mainly proline) as an energy source. Nevertheless, procyclic forms do express a glucose transport system which differs from that found in the bloodstream forms (31,38).In a previous report, we described the cloning and analysis of a developmentally regulated T. brucei gene coding for a putative hexose transporter called THT (for trypanosome hexose transporter) (9). Here we report the characterization of a multigene family encoding two highly related proteins, THT1 and THT2. The genes are all located in a single cluster containing six adjacent copies of the THT1 genes and five copies of the THT2 genes. Expression of these genes during the parasite life cycle is discussed; the hexose transporter * Corresponding author. function of the THT1 gene has been determined by expression in Xenopus oocytes. MATERIALS AND METHODSTrypanosomes. The bloodstream forms of T. brucei brucei EATRO-164 (kindly provided by R. Brun) were obtained from infected rats as described previously (26). Procyclic forms of T. brucei brucei Stib-247 (kindly provided by R. Brun) were cultured at 26°C in a semidefined medium containing 10% fetal calf serum dialyzed by ultrafiltration (Sigma F3260 filter) or undialyzed serum, 3.5 mg of hemin per ml, and 0.5 g of L-proline per liter, with or without 1 g of glucose per liter (3).Construction and screening of genomic libraries. For cloning of THTg.A, total DNA of EATRO-164 was fully digested by EcoRI, and 8-to 25-kb fragments purified by fast protein liquid chrom...

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“…However, if one takes into account that the majority of the label represented metabolites, then the internal concentration of free glucose was well below the external concentration. By contrast, Parsons and Nielsen (16) and Munoz-Antonia et al (13) suggested that in insect-form T. brucei glucose is actively transported and concentrated. Their conclusions were based on the measurement of 2-deoxy-D-glucose rather than glucose itself.…”

Section: Resultsmentioning

confidence: 99%

“…We found that, in the presence of T. brucei cells dissolved in 5.5% perchloric acid, 22.6% of the 2-deoxy-D-glucose-6-phosphate initially present was hydrolyzed after 15 min. In the experiments of Parsons and Nielsen (16), 77% of the label taken up was recovered in the phosphorylated form, but no care was taken to demonstrate that the remaining 23% of unphosphorylated 2-deoxy-D-glucose could not have resulted from hydrolysis of the phosphorylated compound during and after the incubation, thus leading to a gross overestimation of the intracellular concentration of the unphosphorylated form. In similar experiments with D-glucose, there was hardly any incorporation (16).…”

Section: Resultsmentioning

confidence: 99%

“…In the experiments of Parsons and Nielsen (16), 77% of the label taken up was recovered in the phosphorylated form, but no care was taken to demonstrate that the remaining 23% of unphosphorylated 2-deoxy-D-glucose could not have resulted from hydrolysis of the phosphorylated compound during and after the incubation, thus leading to a gross overestimation of the intracellular concentration of the unphosphorylated form. In similar experiments with D-glucose, there was hardly any incorporation (16). Moreover, when we calculate the internal concentration of free unphosphorylated 2-deoxy-D-glucose from the data of Munoz-Antonia et al (13), it remains well below the external glucose concentration.…”

Section: Resultsmentioning

confidence: 99%

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Mutual adjustment of glucose uptake and metabolism in Trypanosoma brucei grown in a chemostat

Kuile

Opperdoes

1992

J Bacteriol

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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-') or a high dilution rate (1 day-'). 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.For optimal adaptation to its environment, an organism needs to obtain the maximum yield from the available energy source. This requires that the individual steps in the metabolic pathway be adjusted to each other in a way that prevents overcapacity in one step relative to the others. Bloodstream trypomastigotes of the protozoan parasite Trypanosoma brucei thrive in the blood and body fluids of their mammalian host and enjoy a stable environment. The uptake and subsequent metabolism of glucose in this organism are an example of an adjustment leading to maximum energy efficiency (26). However, this adjustment should not be considered the general rule in the Trypanosomatidae, because the insect stage of a related species, Leishmania donovani, which in the sandfly midgut is confronted with widely varying conditions, strives for internal homeostasis even at the expense of energy (28). Thus these two very different metabolic strategies may represent two opposing trends: efficient adaptation at the expense of short-term flexibility on the one hand and the ability to rapidly adapt to environmental changes at the expense of energy on the other. Intermediate situations may be found in organisms that encounter only moderate fluctuations in their environmental conditions. An example of the latter type of organism is the procyclic insect stage of T. brucei, under study here, which resides in the midgut of the tsetse fly.T. brucei bloodstream trypomastigotes, when ingested with the blood meal by the tsetse fly, transform in the midgut to the procyclic insect stage. Unlike bloodstream-stage T. brucei, organisms in the insect or procyclic stage are not exclusively dependent on glucose as the carbon and energy source. They are also capable of utilizing proline and other amino acids (7,22). While the blood proteins are being digested by the fly, the parasite is exposed to variations in the concentrations of both glucose and amino acids. Parasite stages in insects may therefore represent an interesting model for the study of adaptations to the...

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Active transport of 2-deoxy-d-glucose in Trypanosoma brucei procyclic forms

Cited by 36 publications

References 21 publications

The Adenosine Analog Tubercidin Inhibits Glycolysis in Trypanosoma brucei as Revealed by an RNA Interference Library

The Adenosine Analog Tubercidin Inhibits Glycolysis in Trypanosoma brucei as Revealed by an RNA Interference Library

Differential regulation of two distinct families of glucose transporter genes in Trypanosoma brucei.

Mutual adjustment of glucose uptake and metabolism in Trypanosoma brucei grown in a chemostat

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