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Bioenergetic consequences of F_oF₁-ATP synthase/ATPase deficiency in two life cycle stages of Trypanosoma brucei.

ABSTRACT: Mitochondrial ATP synthase is a reversible nanomotor synthesizing or hydrolyzing ATP depending on the potential across the membrane in which it is embedded. In the unicellular parasite Trypanosoma brucei, the direction of the complex depends on the life cycle stage of this digenetic parasite: in the midgut of the tsetse fly vector (procyclic form), the F_oF₁-ATP synthase generates ATP by oxidative phosphorylation, whereas in the mammalian bloodstream form, this complex hydrolyzes ATP and maintains mitochondrial membrane potential (ΔΨm). The trypanosome F_oF₁-ATP synthase contains numerous lineage-specific subunits whose roles remain unknown. Here, we seek to elucidate the function of the lineage-specific protein Tb1, the largest membrane-bound subunit. In procyclic form cells, Tb1 silencing resulted in a decrease of F_oF₁-ATP synthase monomers and dimers, rerouting of mitochondrial electron transfer to the alternative oxidase, reduced growth rate and cellular ATP levels, and elevated ΔΨm and total cellular reactive oxygen species levels. In bloodstream form parasites, RNAi silencing of Tb1 by ∼90% resulted in decreased F_oF₁-ATPase monomers and dimers, but it had no apparent effect on growth. The same findings were obtained by silencing of the oligomycin sensitivity-conferring protein, a conserved subunit in T. brucei F_oF₁-ATP synthase. However, as expected, nearly complete Tb1 or oligomycin sensitivity-conferring protein suppression was lethal because of the inability to sustain ΔΨm. The diminishment of F_oF₁-ATPase complexes was further accompanied by a decreased ADP/ATP ratio and reduced oxygen consumption via the alternative oxidase. Our data illuminate the often diametrically opposed bioenergetic consequences of F_oF₁-ATP synthase loss in insect versus mammalian forms of the parasite.

SUBMITTER: Hierro-Yap C

PROVIDER: S-EPMC7949148 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description: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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Bioenergetic consequences of F_oF₁-ATP synthase/ATPase deficiency in two life cycle stages of Trypanosoma brucei.

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Bioenergetic consequences of FoF1-ATP synthase/ATPase deficiency in two life cycle stages of Trypanosoma brucei.

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Bioenergetic consequences of F_oF₁-ATP synthase/ATPase deficiency in two life cycle stages of Trypanosoma brucei.