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. 2000 Mar 1;346(Pt 2):545–552.

Plasmodium falciparum-infected red blood cells depend on a functional glutathione de novo synthesis attributable to an enhanced loss of glutathione.

K Lüersen 1, R D Walter 1, S Müller 1
PMCID: PMC1220884  PMID: 10677377

Abstract

During the erythrocytic cycle, Plasmodium falciparum is highly dependent on an adequate thiol status for its survival. Glutathione reductase as well as de novo synthesis of GSH are responsible for the maintenance of the intracellular GSH level. The first and rate-limiting step of the synthetic pathway is catalysed by gamma-glutamylcysteine synthetase (gamma-GCS). Using L-buthionine-(S, R)-sulphoximine (BSO), a specific inhibitor of the gamma-GCS, we show that the infection with P. falciparum causes drastic changes in the GSH metabolism of red blood cells (RBCs). Infected RBCs lose GSH at a rate 40-fold higher than non-infected RBCs. The de novo synthesis of the tripeptide was found to be essential for parasite survival. GSH depletion by BSO inhibits the development of P. falciparum with an IC(50) of 73 microM. The effect of the drug is abolished by supplementation with GSH or GSH monoethyl ester. Our studies demonstrate that the plasmodicidal effect of the inhibitor BSO does not depend on its specificity towards its target enzyme in the parasite, but on the changed physiological needs for the metabolite GSH in the P. falciparum-infected RBCs. Therefore the depletion of GSH is proposed as a chemotherapeutic strategy for malaria, and gamma-GCS is proposed as a potential drug target.

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Selected References

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  1. Arrick B. A., Griffith O. W., Cerami A. Inhibition of glutathione synthesis as a chemotherapeutic strategy for trypanosomiasis. J Exp Med. 1981 Mar 1;153(3):720–725. doi: 10.1084/jem.153.3.720. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Atamna H., Ginsburg H. Origin of reactive oxygen species in erythrocytes infected with Plasmodium falciparum. Mol Biochem Parasitol. 1993 Oct;61(2):231–241. doi: 10.1016/0166-6851(93)90069-a. [DOI] [PubMed] [Google Scholar]
  3. Atamna H., Ginsburg H. The malaria parasite supplies glutathione to its host cell--investigation of glutathione transport and metabolism in human erythrocytes infected with Plasmodium falciparum. Eur J Biochem. 1997 Dec 15;250(3):670–679. doi: 10.1111/j.1432-1033.1997.00670.x. [DOI] [PubMed] [Google Scholar]
  4. Ayi K., Cappadoro M., Branca M., Turrini F., Arese P. Plasmodium falciparum glutathione metabolism and growth are independent of glutathione system of host erythrocyte. FEBS Lett. 1998 Mar 13;424(3):257–261. doi: 10.1016/s0014-5793(98)00185-9. [DOI] [PubMed] [Google Scholar]
  5. Bailey H. H. L-S,R-buthionine sulfoximine: historical development and clinical issues. Chem Biol Interact. 1998 Apr 24;111-112:239–254. doi: 10.1016/s0009-2797(97)00164-6. [DOI] [PubMed] [Google Scholar]
  6. Bhakdi S., Tranum-Jensen J., Sziegoleit A. Mechanism of membrane damage by streptolysin-O. Infect Immun. 1985 Jan;47(1):52–60. doi: 10.1128/iai.47.1.52-60.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bhattacharya J., Swarup-Mitra S. Reduction in erythrocytic GSH level and stability in Plasmodium vivax malaria. Trans R Soc Trop Med Hyg. 1987;81(1):64–66. doi: 10.1016/0035-9203(87)90285-9. [DOI] [PubMed] [Google Scholar]
  8. Braun Breton C., Rosenberry T. L., Pereira da Silva L. H. Glycolipid anchorage of Plasmodium falciparum surface antigens. Res Immunol. 1990 Oct;141(8):743–755. doi: 10.1016/0923-2494(90)90005-j. [DOI] [PubMed] [Google Scholar]
  9. Butler D., Maurice J., O'Brien C. Time to put malaria control on the global agenda. Nature. 1997 Apr 10;386(6625):535–536. doi: 10.1038/386535a0. [DOI] [PubMed] [Google Scholar]
  10. Cadenas E., Sies H. Oxidative stress: excited oxygen species and enzyme activity. Adv Enzyme Regul. 1985;23:217–237. doi: 10.1016/0065-2571(85)90049-4. [DOI] [PubMed] [Google Scholar]
  11. Clark I. A., Hunt N. H. Evidence for reactive oxygen intermediates causing hemolysis and parasite death in malaria. Infect Immun. 1983 Jan;39(1):1–6. doi: 10.1128/iai.39.1.1-6.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Divo A. A., Geary T. G., Davis N. L., Jensen J. B. Nutritional requirements of Plasmodium falciparum in culture. I. Exogenously supplied dialyzable components necessary for continuous growth. J Protozool. 1985 Feb;32(1):59–64. doi: 10.1111/j.1550-7408.1985.tb03013.x. [DOI] [PubMed] [Google Scholar]
  13. Dubois V. L., Platel D. F., Pauly G., Tribouley-Duret J. Plasmodium berghei: implication of intracellular glutathione and its related enzyme in chloroquine resistance in vivo. Exp Parasitol. 1995 Aug;81(1):117–124. doi: 10.1006/expr.1995.1099. [DOI] [PubMed] [Google Scholar]
  14. Eckman J. R., Eaton J. W. Dependence of plasmodial glutathione metabolism on the host cell. Nature. 1979 Apr 19;278(5706):754–756. doi: 10.1038/278754a0. [DOI] [PubMed] [Google Scholar]
  15. Elford B. C., Pinches R. A. Inducible transport systems in the regulation of parasite growth in malaria-infected red blood cells. Biochem Soc Trans. 1992 Nov;20(4):790–796. doi: 10.1042/bst0200790. [DOI] [PubMed] [Google Scholar]
  16. Fiskerstrand T., Refsum H., Kvalheim G., Ueland P. M. Homocysteine and other thiols in plasma and urine: automated determination and sample stability. Clin Chem. 1993 Feb;39(2):263–271. [PubMed] [Google Scholar]
  17. Fruehauf J. P., Zonis S., al-Bassam M., Kyshtoobayeva A., Dasgupta C., Milovanovic T., Parker R. J., Buzaid A. C. Melanin content and downregulation of glutathione S-transferase contribute to the action of L-buthionine-S-sulfoximine on human melanoma. Chem Biol Interact. 1998 Apr 24;111-112:277–305. doi: 10.1016/s0009-2797(97)00167-1. [DOI] [PubMed] [Google Scholar]
  18. Ginsburg H., Atamna H. The redox status of malaria-infected erythrocytes: an overview with an emphasis on unresolved problems. Parasite. 1994 Mar;1(1):5–13. doi: 10.1051/parasite/1994011005. [DOI] [PubMed] [Google Scholar]
  19. Ginsburg H., Famin O., Zhang J., Krugliak M. Inhibition of glutathione-dependent degradation of heme by chloroquine and amodiaquine as a possible basis for their antimalarial mode of action. Biochem Pharmacol. 1998 Nov 15;56(10):1305–1313. doi: 10.1016/s0006-2952(98)00184-1. [DOI] [PubMed] [Google Scholar]
  20. Griffith O. W. Mechanism of action, metabolism, and toxicity of buthionine sulfoximine and its higher homologs, potent inhibitors of glutathione synthesis. J Biol Chem. 1982 Nov 25;257(22):13704–13712. [PubMed] [Google Scholar]
  21. Huang C. S., Chang L. S., Anderson M. E., Meister A. Catalytic and regulatory properties of the heavy subunit of rat kidney gamma-glutamylcysteine synthetase. J Biol Chem. 1993 Sep 15;268(26):19675–19680. [PubMed] [Google Scholar]
  22. Hunt N. H., Stocker R. Oxidative stress and the redox status of malaria-infected erythrocytes. Blood Cells. 1990;16(2-3):499–530. [PubMed] [Google Scholar]
  23. Hussein A. S., Walter R. D. Purification and characterization of gamma-glutamylcysteine synthetase from Ascaris suum. Mol Biochem Parasitol. 1995 Jun;72(1-2):57–64. doi: 10.1016/0166-6851(94)00064-t. [DOI] [PubMed] [Google Scholar]
  24. Kanaani J., Ginsburg H. Metabolic interconnection between the human malarial parasite Plasmodium falciparum and its host erythrocyte. Regulation of ATP levels by means of an adenylate translocator and adenylate kinase. J Biol Chem. 1989 Feb 25;264(6):3194–3199. [PubMed] [Google Scholar]
  25. Levy E. J., Anderson M. E., Meister A. Transport of glutathione diethyl ester into human cells. Proc Natl Acad Sci U S A. 1993 Oct 1;90(19):9171–9175. doi: 10.1073/pnas.90.19.9171. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Lueder D. V., Phillips M. A. Characterization of Trypanosoma brucei gamma-glutamylcysteine synthetase, an essential enzyme in the biosynthesis of trypanothione (diglutathionylspermidine). J Biol Chem. 1996 Jul 19;271(29):17485–17490. doi: 10.1074/jbc.271.29.17485. [DOI] [PubMed] [Google Scholar]
  27. Lunn G., Dale G. L., Beutler E. Transport accounts for glutathione turnover in human erythrocytes. Blood. 1979 Jul;54(1):238–244. [PubMed] [Google Scholar]
  28. Lüersen K., Walter R. D., Müller S. The putative gamma-glutamylcysteine synthetase from Plasmodium falciparum contains large insertions and a variable tandem repeat. Mol Biochem Parasitol. 1999 Jan 5;98(1):131–142. doi: 10.1016/s0166-6851(98)00161-3. [DOI] [PubMed] [Google Scholar]
  29. Meister A. Glutathione deficiency produced by inhibition of its synthesis, and its reversal; applications in research and therapy. Pharmacol Ther. 1991;51(2):155–194. doi: 10.1016/0163-7258(91)90076-x. [DOI] [PubMed] [Google Scholar]
  30. Meister A. Selective modification of glutathione metabolism. Science. 1983 Apr 29;220(4596):472–477. doi: 10.1126/science.6836290. [DOI] [PubMed] [Google Scholar]
  31. Michelet F., Gueguen R., Leroy P., Wellman M., Nicolas A., Siest G. Blood and plasma glutathione measured in healthy subjects by HPLC: relation to sex, aging, biological variables, and life habits. Clin Chem. 1995 Oct;41(10):1509–1517. [PubMed] [Google Scholar]
  32. Nagel R. L., Roth E. F., Jr Malaria and red cell genetic defects. Blood. 1989 Sep;74(4):1213–1221. [PubMed] [Google Scholar]
  33. Paniker N. V., Beutler E. The effect of methylene blue and diaminodiphenysulfone on red cell reduced glutathione synthesis. J Lab Clin Med. 1972 Oct;80(4):481–487. [PubMed] [Google Scholar]
  34. Pasvol G., Wilson R. J., Smalley M. E., Brown J. Separation of viable schizont-infected red cells of Plasmodium falciparum from human blood. Ann Trop Med Parasitol. 1978 Feb;72(1):87–88. doi: 10.1080/00034983.1978.11719283. [DOI] [PubMed] [Google Scholar]
  35. Spies H. S., Steenkamp D. J. Thiols of intracellular pathogens. Identification of ovothiol A in Leishmania donovani and structural analysis of a novel thiol from Mycobacterium bovis. Eur J Biochem. 1994 Aug 15;224(1):203–213. doi: 10.1111/j.1432-1033.1994.tb20013.x. [DOI] [PubMed] [Google Scholar]
  36. Stocker R., Hunt N. H., Buffinton G. D., Weidemann M. J., Lewis-Hughes P. H., Clark I. A. Oxidative stress and protective mechanisms in erythrocytes in relation to Plasmodium vinckei load. Proc Natl Acad Sci U S A. 1985 Jan;82(2):548–551. doi: 10.1073/pnas.82.2.548. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Tietze F. Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione: applications to mammalian blood and other tissues. Anal Biochem. 1969 Mar;27(3):502–522. doi: 10.1016/0003-2697(69)90064-5. [DOI] [PubMed] [Google Scholar]
  38. Trager W., Jensen J. B. Human malaria parasites in continuous culture. Science. 1976 Aug 20;193(4254):673–675. doi: 10.1126/science.781840. [DOI] [PubMed] [Google Scholar]
  39. Usanga E. A., Luzzatto L. Adaptation of Plasmodium falciparum to glucose 6-phosphate dehydrogenase-deficient host red cells by production of parasite-encoded enzyme. 1985 Feb 28-Mar 6Nature. 313(6005):793–795. doi: 10.1038/313793a0. [DOI] [PubMed] [Google Scholar]
  40. Vennerstrom J. L., Eaton J. W. Oxidants, oxidant drugs, and malaria. J Med Chem. 1988 Jul;31(7):1269–1277. doi: 10.1021/jm00402a001. [DOI] [PubMed] [Google Scholar]
  41. Wozencraft A. O. Damage to malaria-infected erythrocytes following exposure to oxidant-generating systems. Parasitology. 1986 Jun;92(Pt 3):559–567. doi: 10.1017/s0031182000065446. [DOI] [PubMed] [Google Scholar]
  42. Wozencraft A. O., Dockrell H. M., Taverne J., Targett G. A., Playfair J. H. Killing of human malaria parasites by macrophage secretory products. Infect Immun. 1984 Feb;43(2):664–669. doi: 10.1128/iai.43.2.664-669.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Yayon A., Vande Waa J. A., Yayon M., Geary T. G., Jensen J. B. Stage-dependent effects of chloroquine on Plasmodium falciparum in vitro. J Protozool. 1983 Nov;30(4):642–647. doi: 10.1111/j.1550-7408.1983.tb05336.x. [DOI] [PubMed] [Google Scholar]
  44. Zhang Y. A., Hempelmann E., Schirmer R. H. Glutathione reductase inhibitors as potential antimalarial drugs. Effects of nitrosoureas on Plasmodium falciparum in vitro. Biochem Pharmacol. 1988 Mar 1;37(5):855–860. doi: 10.1016/0006-2952(88)90172-4. [DOI] [PubMed] [Google Scholar]
  45. Zhang Y., König I., Schirmer R. H. Glutathione reductase-deficient erythrocytes as host cells of malarial parasites. Biochem Pharmacol. 1988 Mar 1;37(5):861–865. doi: 10.1016/0006-2952(88)90173-6. [DOI] [PubMed] [Google Scholar]
  46. Zängerle L., Cuénod M., Winterhalter K. H., Do K. Q. Screening of thiol compounds: depolarization-induced release of glutathione and cysteine from rat brain slices. J Neurochem. 1992 Jul;59(1):181–189. doi: 10.1111/j.1471-4159.1992.tb08889.x. [DOI] [PubMed] [Google Scholar]

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