Effect of inhibitors of electron transport and oxidative phosphorylation on trypanosoma cruzi respiration and growth

Antimycin A and 2-heptyl-4-hydroxyquinoline N-oxide, two specific inhibitors of the b-c₁ segment of the respiratory chain, affected the respiration of Trypanosoma cruzi epimastigote forms. The half-maximum inhibitory concentration were about 0.05 and 0.04 μg/mg cells (dry wt.), respectively. The maximum effect of antimycin (about 80% inhibition of respiration) was at about 0.1 μg antimycin/mg cells. Differential spectrophotometry of T. cruzi epimastigotes in the presence of antimycin, cyanide (or sulfide) and uncouplers, revealed the presence of functional cytochromes aa₃, b and c₅₅₈. In the stationary growth phase respiration by T. cruzi was completely inhibited by cyanide and effectively ihibited by sulfide, but in the expontential growth phase respiration was about 20% insensitive to 5 mM cynaide. Cyanide- and antimycin-insensitive respiration was completely inhibited by salicylhydroxamic acid (2 mM).Antimycin inhibited the operation of the tricarboxylic acids cycle in T. cruzi, as shown by the lesser production of ¹⁴CO₂ and by the modification of ¹⁴C distribution in epimastigotes incubated with [1-¹⁴C]glucose, [2-¹⁴C]acetate or NaH¹⁴CO₃. The inhibition of electron transport by antimycin increased the rate of the fumarate reductase reaction, an alternative electron pathway for the oxidation of reduced pyridine nucleotides.Addition of carbonyl cyanide 3-chlorophenylhydrazone to epimastigotes increased the rate of respiration and promoted the oxidation of reduced cytochrome b components, thus showing that these components are subjects to respiratory (acceptor) control. Pentachlorhenol similarly affected the cytochrome b redox level but did not modify the rate of respiration. The uncouplers released N,N′-dicyclohexycarbodiimide inhibition of respiration, and uncouplers and cyanide significantly decreased the ATP level in epimastigotes. The combined effects of the assayed inhibitors on respiration, cytochrome b redox level, ATP content and energy charge confirmed the operation of oxidative phosphorylation in T. cruzi epimastigotes. Antimycin, uncouplers and N,N′-dicyclohexylcarbodiimide inhibited growth of T. cruzi, thus proving the essential role of oxidative phosphorylation for the parasite.