Membrane-related processes and overall energy metabolism inTrypanosoma brucei and other kinetoplastid species

Kuile, Benno H. ter

doi:10.1007/bf00763065

Cited by 17 publications

(7 citation statements)

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“…Given the functional uniqueness of the mitochondrial F 1 F 0 -ATPase in T. brucei BFs relative to the ATPase in mammalian host cells, the inhibition of ATP hydrolysis by DB75 is likely to represent a mechanism of killing that is parasite selective. Another major distinction of BF trypanosomes relative to mammalian cells or procyclics is that the mitochondria of BFs lack cytochromes and respiration is entirely dependent on the trypanosome alternative oxidase (TAO) system, consisting of D-glycerol-3-phosphate dehydrogenase and a terminal oxidase (3,7,9,42). Therefore, inhibitors of TAO activity in vitro are expected to reduce respiration in BFs.…”

Section: Discussionmentioning

confidence: 99%

The Mitochondrion Is a Site of Trypanocidal Action of the Aromatic Diamidine DB75 in Bloodstream Forms of Trypanosoma brucei

Lanteri¹,

Tidwell²,

Meshnick

2008

Antimicrob Agents Chemother

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Human African trypanosomiasis (HAT) is a fatal tropical disease caused by infection with protozoans of the speciesNew therapies are desperately needed for the treatment of human African trypanosomiasis (HAT). This reemerging tropical disease afflicts as many as 500,000 people (2, 5). Current drugs, such as pentamidine, suramin, melarsoprol, eflornithine, and nifurtimox, are often associated with deleterious side effects and cannot be administered orally (5,16,19,34).A promising new therapy, DB289 [2,5-bis-(4-amidinophenyl)-furan bis-O-methylamidoxime], is undergoing phase III clinical trials for registration as the first orally administered drug for early-stage HAT (19). The oral prodrug DB289 is metabolically converted to the trypanocidal agent DB75 [2,5-bis(4-amidinophenyl)furan] (6, 50, 51). DB75 is active against Trypanosoma spp. in vitro (11,12) and is a structural analogue of the aromatic diamidine drug pentamidine.DB75 and pentamidine are antimicrobial diamidine-type compounds that are active against various fungal and protozoal infections. However, the mechanism through which diamidine drugs exert their activity is still not entirely known and continues to be a focus of extensive research (46). We previously investigated the mechanisms of action of DB75 and pentamidine in yeast cells and found that both drugs appear to disrupt the mitochondrial function in Saccharomyces cerevisiae by collapsing the mitochondrial membrane potential (⌿ m ) and inhibiting mitochondrial respiration (24). We also showed that DB75 and pentamidine inhibit oxidative phosphorylation in isolated rat liver mitochondria (24), which is consistent with the results attained with pentamidine in a previous study (31).The purpose of this investigation was to gain insight into the mechanism of trypanocidal action of DB75. In particular, we investigated if DB75 acts against Trypanosoma brucei bloodstream forms (BFs) by targeting the mitochondrion. MATERIALS AND METHODSChemicals. DB75 [2,5-bis(4-amidinophenyl)furan dihydrochloride] was obtained from David Boykin at Georgia State University, Atlanta, and pentamidine isethionate [1,5-di(4-amidinophenoxy)pentane isethionate] was prepared by LyphoMed, Inc. (Melrose Park, IL). All other chemicals were of the highest quality available and were from Sigma-Aldrich (St. Louis, MO), unless otherwise stated.Collection and isolation of trypanosomes from infected blood. Male SpragueDawley rats and Swiss-Webster mice (Charles River Laboratories, Cambridge, MA) were infected by intraperitoneal injection of approximately 2 ϫ 10 6 and 5 ϫ 10 5 BFs of Trypanosoma brucei brucei strain 427 cells, respectively. Infected blood was collected through cardiac puncture at the time of peak parasitemia (usually day 5 postinfection for rats and day 4 postinfection for mice). The blood was centrifuged in heparinized tubes at 2,200 ϫ g for 10 min at 4°C, and the buffy coat was removed and diluted 1:3 in phosphate saline glucose buffer, pH 8.0. The trypanosomes were purified from the blood by the use of DE52 anion-exchange...

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Section: Discussionmentioning

confidence: 99%

The Mitochondrion Is a Site of Trypanocidal Action of the Aromatic Diamidine DB75 in Bloodstream Forms of Trypanosoma brucei

Lanteri¹,

Tidwell²,

Meshnick

2008

Antimicrob Agents Chemother

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“…5 mM), and energy generation by the bloodstream form occurs exclusively via glycolysis. Glucose enters bloodstream trypomastigotes through a facilitateddiffusion transporter independent of a proton motive force, and glycolysis takes place in a unique organelle, the glycosome [2][3][4][5][6][7]. The end-product of glycolysis in bloodstream trypomastigotes is pyruvate, which is excreted by a facilitated-diffusion pyruvate Abbreviations used : BCECF, 2h,7h-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein ; α-CHC, α-cyanohydroxycinnamic acid ; DCCD, N,Nh-dicyclohexylcarbodi-imide ; DES, diethylstilboestrol ; EIPA, 5-(N-ethyl)-N-isopropylamiloride ; H 2 DIDS, 4,4h-di-isothiocyanatodihydrostilbene-2,2h-disulphonic acid ; LDH, lactate dehydrogenase ; NEM, N-ethylmaleimide ; pH e , external pH ; pH i , intracellular pH.…”

Section: Introductionmentioning

confidence: 99%

A pyruvate‒proton symport and an H+-ATPase regulate the intracellular pH of Trypanosoma brucei at different stages of its life cycle

Vanderheyden

Wong

Docampo³

2000

Biochem. J.

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Regulation of intracellular pH (pH(i)) and H(+) efflux were investigated in Trypanosoma brucei bloodstream and procyclic trypomastigotes using the fluorescent dyes 2', 7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) acetoxymethyl ester and free BCECF respectively. pH(i) in bloodstream and procyclic trypomastigotes was 7.47+/-0.06 and 7. 53+/-0.07 respectively. Differences in the mechanisms for the regulation of pH(i) were noted between bloodstream and procyclic forms. Procyclic trypomastigotes maintained their pH(i) at neutral over a wide range of external pH values from 6 to 8, and in the absence of K(+) or Na(+). The H(+)-ATPase inhibitors N, N'-dicyclohexylcarbodi-imide (DCCD), diethylstilboestrol and N-ethylmaleimide substantially decreased the steady-state pH(i) and inhibited its recovery from acidification. The rate of H(+) efflux in these forms was determined to be 62+/-6.5 nmol/min per mg of protein, and was substantially decreased by H(+)-ATPase inhibitors. The data support the presence of an H(+)-ATPase as the major regulator of pH(i) in procyclic trypomastigotes. In contrast, bloodstream trypomastigotes were unable to maintain a neutral pH under acidic conditions, and their steady-state pH(i) and recovery from acidification were unaffected by H(+)-ATPase inhibitors, except for DCCD (100 microM). Their steady-state pH(i) was markedly decreased in glucose-free buffer or by >/=10 mM pyruvate, whereas procyclic trypomastigotes were unaffected by similar treatments. The rate of H(+) efflux in bloodstream trypomastigotes was 534+/-38 nmol/min per mg of protein, and was decreased in the absence of glucose and by the addition of pyruvate or DCCD. Pyruvate efflux in these forms was calculated to be 499+/-34 nmol/min per mg of protein, and was significantly inhibited by DCCD, 4, 4'-di-isothiocyanatodihydrostilbene-2,2'-disulphonic acid and alpha-cyanohydroxycinnamic acid. The pyruvate analogues beta-hydroxypyruvate, 3-bromopyruvate, 3-oxoglutarate, oxaloacetate, 3-oxoisovalerate and 3-oxoisohexanoate significantly decreased pH(i), as well as proton and pyruvate efflux, whereas lactate had only a small effect, and no effect was observed with citrate or fumarate. The inhibition by pyruvate analogues of pyruvate efflux, proton efflux and acidification of pH(i) supports the hypothesis that pyruvate efflux is accompanied by proton efflux and that this is the major pH(i) control mechanism in bloodstream forms. Inhibition by H(+)-ATPase inhibitors of residual H(+) efflux in the absence of glucose or in the presence of high extracellular pyruvate indicates a minor role for H(+)-ATPase(s) in control of pH(i) in bloodstream forms.

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“…Glucose enters bloodstream-form T. brucei by facilitated diffusion (21,22). Many inhibitors of this process are available (13,15).…”

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confidence: 99%

Contribution of glucose transport to the control of the glycolytic flux in Trypanosoma brucei

Bakker

Walsh

Kuile

et al. 1999

Proc. Natl. Acad. Sci. U.S.A.

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The rate of glucose transport across the plasma membrane of the bloodstream form of Trypanosoma brucei was modulated by titration of the hexose transporter with the inhibitor phloretin, and the effect on the glycolytic f lux was measured. A rapid glucose uptake assay was developed to measure the transport activity independently of the glycolytic f lux. Phloretin proved a competitive inhibitor. When the effect of the intracellular glucose concentration on the inhibition was taken into account, the f lux control coefficient of the glucose transporter was between 0.3 and 0.5 at 5 mM glucose. Because the f lux control coefficients of all steps in a metabolic pathway sum to 1, this result proves that glucose transport is not the rate-limiting step of trypanosome glycolysis. Under physiological conditions, transport shares the control with other steps. At glucose concentrations much lower than physiological, the glucose carrier assumed all control, in close agreement with model predictions.

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Membrane-related processes and overall energy metabolism inTrypanosoma brucei and other kinetoplastid species

Cited by 17 publications

References 39 publications

The Mitochondrion Is a Site of Trypanocidal Action of the Aromatic Diamidine DB75 in Bloodstream Forms of Trypanosoma brucei

The Mitochondrion Is a Site of Trypanocidal Action of the Aromatic Diamidine DB75 in Bloodstream Forms of Trypanosoma brucei

A pyruvate‒proton symport and an H+-ATPase regulate the intracellular pH of Trypanosoma brucei at different stages of its life cycle

Contribution of glucose transport to the control of the glycolytic flux in Trypanosoma brucei

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