Acta Parasitologica

, Volume 59, Issue 1, pp 11–16 | Cite as

Generation of adenosine tri-phosphate in Leishmania donovani amastigote forms

  • Subhasish Mondal
  • Jay Jyoti Roy
  • Tanmoy Bera
Original Paper


Leishmania, the causative agent of various forms of leishmaniasis, is the significant cause of morbidity and mortality. Regarding energy metabolism, which is an essential factor for the survival, parasites adapt to the environment under low oxygen tension in the host using metabolic systems which are very different from that of the host mammals. We carried out the study of susceptibilities to different inhibitors of mitochondrial electron transport chain and studies on substrate level phosphorylation in wild-type L. donovani. The amastigote forms of L. donovani are independent on oxidative phosphorylation for ATP production. Indeed, its cell growth was not inhibited by excess oligomycin and dicyclohexylcarbodiimide, which are the most specific inhibitors of the mitochondrial Fo/F1-ATP synthase. In contrast, mitochondrial complex I inhibitor rotenone and complex III inhibitor antimycin A inhibited amastigote cell growth, suggesting the role of complex I and complex III in cell survival. Complex II appeared to have no role in cell survival. To further investigate the site of ATP production, we studied the substrate level phosphorylation, which was involved in the synthesis of ATP. Succinate-pyruvate couple showed the highest substrate level phosphorylation in amastigotes whereas NADH-fumarate and NADH-pyruvate couples failed to produce ATP. In contrast, NADPH-fumarate showed the highest rate of ATP formation in promastigotes. Therefore, we can conclude that substrate level phosphorylation is essential for the survival of amastigote forms of Leishmania donovani.


ATP Leishmania amastigote promastigote substrate level phosphorylation oligomycin 


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  1. Assaily W., Benchimol S. 2006. Differential utilization of two ATPgenerating pathways is regulated by p53. Cancer Cell, 10, 4–6. DOI: 10.1016/j.ccr.2006.06.014.PubMedCrossRefGoogle Scholar
  2. Barak E., Amin-Spector S., Gerliak E., Goyard S., Holland N., Zilberstein D. 2005. Differentiation of Leishmania donovani in host free system: analysis of signal perception and response. Molecular and Biochemical Parasitology, 141, 99–108. DOI: 10.1016/j.molbiopara.2005.02.004.PubMedCrossRefGoogle Scholar
  3. Bente M., Harder S., Wiesgigl M., Heukeshoven J., Gelhous C., Kranse E., Clos J., Bruchhans I. 2003. Developmentally induced changes of the proteome in the protozoan parasite Leishmania donovani. Proteomics, 3, 1811–1829. DOI: 10.1002/pmic.20030046.PubMedCrossRefGoogle Scholar
  4. Chakraborty B., Biswas S., Mondal S., Bera T. 2010. Stage specific developmental changes in the mitochondrial and surface membrane associated redox systems of Leishmania donovani promastigote and amastigote. Biochemistry (Moscow), 75, 494–504. DOI: 10.1134/S0006297910040140.CrossRefGoogle Scholar
  5. Chappuis F., Sundar S., Haihe A., Ghalib H., Raijal S. 2007. Visceral Leishmaniasis: What are the needs for diagnosis, treatment and control ? Nature Reviews Microbiology, 5, 873–882. DOI: 10.1038/nrmicro1748.PubMedCrossRefGoogle Scholar
  6. Coustou V., Bisteiro S., Brian M., Diolez P., Bouchaud V., Voisin P., Michels P.A.M., Canioni P., Beltz T., Bringaud F. 2003. ATP generation in the Trypanosoma brucei procyclic form: Cytosolic substrate level phosphorylation is essential, but not oxidative phosphorylation. Journal of Biological Chemistry, 278, 49625–49635. DOI 10.1074/jbc.M307872200.PubMedCrossRefGoogle Scholar
  7. Coombs G.H., Croft J.A., Hart D.T. 1982. A comparative study of Leishmania mexicana amastigotes and promastigotes: enzyme activities and subcellular locations. Molecular and Biochemical Parasitology, 5, 199–211. DOI: 10.1016/0166-6851(82) 90021-4.PubMedCrossRefGoogle Scholar
  8. Debrabant A., Joshi M.B., Pimenta P.F., Dwyer D. 2004. Generation of Leishmania donovani axenic amastigotes: their growth and biological characteristics. International Journal of Parasitology, 34, 205–217. DOI: 10.1016/j.ijpara.2003.10.011.PubMedCrossRefGoogle Scholar
  9. Ephros M., Bitnun A., Shaked P., Waldman E., Zinberstein D. 1999. Stage-specific activity of pentavalent antimony against Leishmania donovani axenic amastigotes. Antimicrobial Agents and Chemotherapy, 43, 278–282. DOI: 0066-4804/99.PubMedCentralPubMedGoogle Scholar
  10. Gornall A.G., Bardawill C.J., David M.M. 1949. Determination of serum proteins by means of the biuret reaction. Journal of Biological Chemistry, 177, 751–766.PubMedGoogle Scholar
  11. Gupta N., Goyal N., Singha U.K., Bhakuni V., Roy R., Rastogi A.K. 1999. Characterization of intracellular metabolites of axenic amastigotes of Leishmania donovani by 1H NMR spectroscopy. Acta Tropica, 73, 121–133. DOI: 10.1016/S0001-706X(99)00020-0.PubMedCrossRefGoogle Scholar
  12. Hassan H.F., Coombs G.H. 1985. Leishmania mexicana, purine metabolizing enzymes of amastigotes and promastigotes. Experimental Parasitology, 59, 139–150. DOI: 10.1016/0014-4894 (85)90066-9.PubMedCrossRefGoogle Scholar
  13. Huber W., Koella J.C. 1993. A comparision of the methods of estimating EC50 in studies of drug resistance of malaria parasites. Acta Tropica, 55, 257–261. DOI: 10.1016/0001-706X(93) 90083-N.PubMedCrossRefGoogle Scholar
  14. James P.E., Grinberg O.Y., Swartz H.M. 1998. Superoxide production by phagocytosing macrophages in relation to the intracellular distribution of oxygen. Journal of Leukocyte Biology, 64, 78–84.PubMedGoogle Scholar
  15. Katwa S.D., Katyare S.S. 2003. A simplified method for inorganic phosphate determination and its application for phosphate analysis in enzyme assays. Analytical Biochemistry, 323, 180–187. DOI: 10.1016/j.ab.2003.08.024.CrossRefGoogle Scholar
  16. Lemorse S.L., Sereno D., Danlouede S., Veyret B., Brajon N., Vincendeau P. 1997. Leishmania spp.: nitric oxide-mediated metabolic inhibition of promastigotes and axenically grown amastigote forms. Experimental Parasitology, 86, 58–68. DOI: 10.1006/expr.1997.4151.CrossRefGoogle Scholar
  17. Martin E., Simon M.W., Schaefer F.W., Mukkada A.J. 1976. Enzymes of carbohydrate metabolism in four human species of Leishmania: a comparative survey. Journal of Protozoology, 23, 600–607. DOI: 10.1111/j.1550-7408.1976.tb03850.PubMedCrossRefGoogle Scholar
  18. Mattock N.M., Peters W. 1975. The experimental chemotherapy of leishmaniasis. II. The activity in tissue culture of some antiparasitic and antimicrobial compounds in clinical use. Annals of Tropical Medicine and Parasitology, 69, 359–371.PubMedGoogle Scholar
  19. Mc Conville M.J., de Souza D., Saunders E., Likic V.A. Naderer T. 2007. Living in a phagolysosome; metabolism of Leishmania amastigotes. Trends in Parasitology, 23, 368–375. DOI: 10.1016/ Scholar
  20. Michels P.A.M., Michels J.P.J., Boonstra J., Konings W.N. 1979. Generation of an electropotential proton gradient in bacteria by the excretion of metabolic end products. FEMS Microbiology Letters, 5, 357–364. DOI: 10.1111/j.1574-6968.1979.tb03339.CrossRefGoogle Scholar
  21. Naderer T., Mc Conville M.J. 2008. The Leishmania macrophage interaction: a metabolic perspective. Cellular Microbiology, 10, 301–308. DOI: 10.1111/j.1462-5822.2007.01096.PubMedCrossRefGoogle Scholar
  22. Peters W., Trotter E.R., Robinson B.L. 1980. The experimental chemotherapy of leishmaniasis, VII. Drug responses of L. major and L. mexicana amazonensis, with an analysis of promising chemical leads to new antileishmanial agents. Annals of Tropical Medicine and Parasitology, 74, 321–335.PubMedGoogle Scholar
  23. Rainey P.M., Spithill T.W., Mc Mahon-Pratt D., Pan A.A. 1991. Biochemical molecular characterization of Leishmania pefanoi amastigotes in continuos culture. Molecular and Biochemical Parasitology, 49, 111–118. DOI: 10.1016/0166-6851(91)90134-R.PubMedCrossRefGoogle Scholar
  24. Rainey P.M., MacKenzie N.E. 1991. A carbon-13 nuclear magnetic resonance analysis of the products of glucose metabolism in Leishmania pifanoi amastigotes and promastigotes. Molecular and Biochemical Parasitology, 45, 307–315. DOI: 10.1016/0166-6851(91)90099-R.PubMedCrossRefGoogle Scholar
  25. Rivas L., Chang L.P. 1983. Intraparasitophorous vacuolar pH of Leishmania mexicana infected macrophages. Biological Bulletin, 165, 536–537.Google Scholar
  26. Rudzinska M.A., Alesandro P.A.D., Trager W. 1964. The fine structure of Leishmania donovani and the role of the kinetoplast in the leishmani-leptomonad transformation. Journal of Protozoology, 11, 166–191. DOI: 10.1111/j.1550-7408.1964.tb01739.PubMedCrossRefGoogle Scholar
  27. Saar Y., Ransfold A., Waldman E., Mazareb S., Amin-Spector S., Plumblee J., Turco S.J., Zilberstein D. 1998. Characterization of developmentally regulated activities in axenic amastigotes of Leishmania donovani. Molecular and Biochemical Parasitology, 95, 9–20. DOI: 10.1016/S0166-6851(98)00062-0.PubMedCrossRefGoogle Scholar
  28. Sereno D., Lemesre J.L. 1997. Axenically cultured amastigote forms as an in vitro model for investigation of antileishmanial agents. Antimicrobial Agents and Chemotherapy, 41, 972–976. DOI: 0066-4804/97.PubMedCentralPubMedGoogle Scholar
  29. Singh A.K., Mukhopadhyay C., Biswas S., Singh V.K., Mukhopadhyay C.K. 2012. Intracellular pathogen Leishmania donovani activates hypoxia inducible factor-1 by dual mechanism for survival advantage within macrophage. Plos One, 7, e38489. DOI: 10.1371/journal.pone.0038489.PubMedCentralPubMedCrossRefGoogle Scholar
  30. Tielens A.G., Van Hellemond J.J. 1998. The electron transport chain in anaerobically functioning eukaryotes. Biochimica et Biophysica Acta (Bioenergetics), 1365, 71–78. DOI: 10.1016/S0005-2728(98)00045-0.CrossRefGoogle Scholar
  31. Van Hellemond J.J., Van der Klei A., van Weelden S.W., Tielens A.G. 2003. Biochemical and evolutionary aspects of anaerobically functioning mitochondria. Philosophical Transactions of the Royal Society B: Biological Science, 358, 205–213. DOI: 10.1098/rstb.2002.1182.CrossRefGoogle Scholar
  32. Wennberg E., Weiss L. 1969. The structure of the spleen and hemolysis. Annual Review of Medicine, 20, 29–40. DOI: 10.1146/ Scholar
  33. Zilberstein D., Shapira M. 1994. The role of pH and temperature in the development of Leishmania parasites. Annual Review of Microbiology, 48, 449–470. DOI: 10.1146/annurev.mi.48.100194.002313.PubMedCrossRefGoogle Scholar

Copyright information

© Versita Warsaw and Springer-Verlag Wien 2014

Authors and Affiliations

  1. 1.Division of Medicinal Biochemistry, Department of Pharmaceutical TechnologyJadavpur UniversityKolkataIndia

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