Terminal oxidases in the trypanosomatid Trypanosoma cruzi
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Abstract:
Titration of Trypanosoma cruzi respiration with cyanide, with results treated as Dixon plots, indicated the presence of several terminal oxidases. The inhibitions obtained at low cyanide concentrations (0-300 microM), taken together with cyanide effects on cytochrome aa3-deficient, dyskinetoplastic epimastigotes, supported cytochrome aa3 as T. cruzi main terminal oxidase. By increasing cyanide concentration to 1.0 mM, two alternative terminal oxidases could be detected. One of these was active in both kinetoplastic and dyskinetoplastic (cytochrome aa3-deficient) epimastigotes, and azide- and antimycin-insensitive. Complementary cytochrome studies with intact epimastigotes and mitochondrial membranes revealed the presence of cytochromes aa3, b, c558, o and possibly d, as components of the parasite electron transport system. Fractionation studies demonstrated that both o and d were bound to the mitochondrial membrane. Reduction by endogenous substrates and complex formation with cyanide supported cytochrome o as alternative terminal oxidase. EB-cultured, dyskinetoplastic epimastigotes showed the same respiration rate as the kinetoplastic cells, despite the significant decrease of cytochrome aa3, thus indicating adaptive mechanisms that determine the expression of alternative oxidases, whenever the main terminal activity is depressed.
Insights
Trypanosoma cruzi respiration involves multiple terminal oxidases. Cytochrome aa3 is the primary oxidase, but alternative oxidases ensure respiration when cytochrome aa3 is deficient.
Area of Science:
- Biochemistry
- Parasitology
- Molecular Biology
Background:
- Trypanosoma cruzi, the causative agent of Chagas disease, relies on mitochondrial respiration.
- Understanding the parasite's electron transport system is crucial for developing targeted therapies.
Purpose of the Study:
- To identify and characterize the terminal oxidases involved in Trypanosoma cruzi respiration.
- To investigate the role of alternative oxidases in parasite survival.
Main Methods:
- Cyanide titration and Dixon plot analysis of Trypanosoma cruzi respiration.
- Enzyme inhibition studies using cyanide, azide, and antimycin.
- Cytochrome difference spectroscopy on intact epimastigotes and mitochondrial membranes.
- Subcellular fractionation to localize oxidases.
Main Results:
- Cytochrome aa3 was identified as the main terminal oxidase in Trypanosoma cruzi.
- Two alternative terminal oxidases were detected at higher cyanide concentrations.
- One alternative oxidase was insensitive to azide and antimycin, suggesting a novel pathway.
- Cytochromes b, c558, o, and possibly d were identified in the electron transport system.
- Cytochrome o was supported as an alternative terminal oxidase.
- Dyskinetoplastic epimastigotes maintained respiration rates despite cytochrome aa3 deficiency, indicating adaptive oxidase expression.
Conclusions:
- Trypanosoma cruzi possesses a complex respiratory system with multiple terminal oxidases.
- Alternative oxidases play a significant role in maintaining parasite respiration, especially under conditions of cytochrome aa3 deficiency.
- These findings offer potential targets for anti- Trypanosoma cruzi drug development.