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

Abstract

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.

A Vacuolar-type H+-Pyrophosphatase Governs Maintenance of Functional Acidocalcisomes and Growth of the Insect and Mammalian Forms of Trypanosoma brucei

“…Therefore, we analyzed the steady-state pH i of these mutants under different external pH (pH e ). In normal cells, the pH i remained close to 7.1 in the pH e ranges from 6.5 to 8 as reported previously (30,44). However, while the pH i of the induced mutant cells was maintained at acidic pH e , it increased almost 0.2 pH unit at basic pH e (Fig.…”

“…To further investigate the role of the V-H ϩ -PPase in pH i regulation, procyclic trypomastigotes were acidified to a pH of ϳ6.0 using the NH 4 Cl prepulse technique (44). Fig.…”

Effect of 3-Bromopyruvate and Atovaquone on Infection during In Vitro Interaction of Toxoplasma gondii and LLC-MK2 Cells

“…In other protozoans, such as Trypanosoma brucei, the inhibitory effects of 3-BrPA on parasite motility and the efflux of pyruvate resulted in parasite death due to a blockade of the enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (19,20). It has been reported that this enzyme plays a significant role in the glycolytic flux of protozoa, such as Leishmania mexicana, Plasmodium falciparum, and T. brucei (32,33).…”

“…In protozoa such as Trypanosoma brucei, 3-BrPA has inhibitory effects on (i) the motility of the parasite at 13.4 mM and (ii) the efflux of pyruvate at 5.6 mM due to blockage of GAPDH, leading the reduced parasite viability (19,20). These data prompted us to study the activity of 3-BrPA on the intracellular development of T. gondii in LCC-MK2 cells.…”

Molecular Identification and Characterization of an Essential Pyruvate Transporter from Trypanosoma brucei

“…The presence of pyruvate transporters in the plasma membrane of T. brucei has been established by biochemical studies demonstrating their physiological importance (6,7,13,14). However, the molecular identification of these important transporters has eluded discovery over an extended period of time.…”