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Leishmania donovani: In vitro culture and [1H] NMR characterization of amastigote-like forms

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Abstract

WhenLeishmania donovani promastigote forms, were cultured in TC-199 medium at 28°C and subsequently incubated at 38°C, they turned into aflagellate (amastogote-like) forms. A return of the incubation-culture temperature to 28°C these amastigote-like forms to revert to promastigotes. The amastigotes obtained by heat-shock, were viable and retained antigenic capacity being recognized by the sera of naturally infected patients. These forms, remained also capable of multiplying inside the J-774A. 1 macrophages. When the amastigote-like forms are kept in culture at 38°C retained their rounded appearance and their biological characteristics for more than 3 months subculturing every 6 days. These amastigote-like forms, when used for subcultures at 28°C, transformed into promastigotes capable of multiplying, as flagellate forms. The amastigotelike forms obtainedin vitro can be used in biochemical studies related to chemotherapy and immunology studies, as part of an effort to combat this parasite. The end-products of of glycolysis were studied in both the amastigote-like and promastigote forms ofL. donovani, by proton magnetic resonance analysis of the culture media. Alanine, succinate, and acetate, were predominant, and to a lesser extent pyruvate, glycine and D-lactate. Our results suggest that both forms ofLeishmania use different biochemical strategies to obtain their energy.

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References

  1. Alexander J, Vickerman K: Fusion of host cell secondary lysosomes with parasitophorous vacuoles ofLeishmania mexicana-infected macrophages. J Protoz 22: 502–508, 1975

    PubMed  Google Scholar 

  2. Chang KP, Dwyer DM: Multiplication of a human parasite (Leishmania donovani) in phagolysosomes of hamster macrophagesin vitro. Science 193: 678–680, 1976

    PubMed  Google Scholar 

  3. Pan AA:Leishmania mexicana: Serial cultivation of intracellular stages in a cell-free medium. Exp Parasit 58: 72–80, 1984

    PubMed  Google Scholar 

  4. Smejkal RM, Geller R, Hansen BD, Olenick JG: A two-dimensional gel electrophoresis analysis of the conversion ofLeishmania brasiliensis panamensis promastigotes to amastigotes forms. Fed Proc 43: 1791–1795, 1984

    Google Scholar 

  5. Lawrence F, Robert-Gero M: Induction of heat shock and stress proteins in promastigotes of threeLeishmania species. Proc Natl Acad Science 82: 4414–4417, 1985

    Google Scholar 

  6. Darling TN, Blum JJ:In vitro reversible transformation ofLeishmania brasiliensis panamensis between promastigotes and ellipsoidal forms. J Protoz 34: 166–168, 1987

    Google Scholar 

  7. Stinson S, Sommer JR, Blum JJ: Morphology ofLeishmania brasiliensis: Change during reversible heat-induced transformation from promastigotes to an ellipsoidal form. J Parasit 75: 431–440, 1989

    PubMed  Google Scholar 

  8. Pan AA:Leishmania mexicana pifanoi: Analysis of antigenic relationships between promastigotes and amastigotes by gel diffusion, immunoelectrophoresis, and immunoprecipitation. J Protoz 33: 192–197, 1986

    Google Scholar 

  9. Smejkal RM, Wolff R, Olenick JG:Leishmania brasiliensis panamensis: Increased infectivity resulting from heat shock. Exp Parasit 65: 1–9, 1988

    PubMed  Google Scholar 

  10. Eperon S, McMahon-Pratt D: Extracellular cultivation and morphological characterization of amastigote like forms ofLeishmania panamensis andLeishmania brasiliensis. J Protoz 36: 502–510, 1988a

    Google Scholar 

  11. Eperon S, McMahon-Pratt D: Extracellular amastigote-like forms ofLeishmania panamensis andLeishmania brasiliensis. Stage- and species-specific monoclonal antibodies. J Protoz 36: 510–518, 1988b

    Google Scholar 

  12. Doyle PS, Engel JC, Pimenta PFP, Da Silva PP, Dwyer DM:Leishmania donovani: Long-term Culture of Axenic Amastigotes at 37°C. Exp Parasit 73: 326–334, 1991

    PubMed  Google Scholar 

  13. Gamarro F, Osuna A, Castanys S, Perez-Lopez MI, Ruiz-Perez LM: Isolation and purification of amastigotes ofTrypanosoma cruzi from cultured Vero cells. Zeitschrift für Parasitenkunden 71: 15–17, 1985

    Google Scholar 

  14. Hames BD: An introduction to polyacrylamide gel electrophoresis. In Gel electrophoresis of proteins: a practical approach, BD Hames and D Rickwood (eds), IRL Press, London, Washington DC, pp 6–14, 1981

    Google Scholar 

  15. Bergmeyer HU: Methods of Enzymatic Analysis, 2nd edn., Academic Press, New York, 1974

    Google Scholar 

  16. Molyneux, DH, Killick-Kendrick R: Morphology, ultrastructure and life cycles. Academic Press, London, pp 121–176, 1987

    Google Scholar 

  17. Kar K, Mukerji K, Naskar K, Bhattacharya A, Ghosh DK:Leishmania donovani: A chemically defined medium suitable for cultivation and cloning of promastigotes and transformation of amastigotes to promastigotes. J Protoz 37 (4): 277–279, 1990

    Google Scholar 

  18. Chang KP, Hendricks LD: Laboratory cultivation and maintenance ofLeishmania. In KP Chang and RS Bray (eds) Elsevier Science Publishers B.V. (Biomedical Division). New York, pp 213–244, 1976

    Google Scholar 

  19. Zilberstein D, Blumenfeld N, Liveanu V, Gepstein A, Jaffe CL: Growth at acidic pH induces an amastigote stage-specific protein inLeishmania promastigotes. Mol Biochem Parasit 45: 175–178, 1991

    Google Scholar 

  20. Shapira M, Mcewab JC, Jaffe CL: Temperature effects on molecular processes which lead to stage differentiation inLeishmania. EMBO Journal 7: 2895–2901, 1988

    PubMed  Google Scholar 

  21. Van Der Ploeg LHT, Giannini SH, Cantor CR: Heat shock genes: Regulatory role for differentiation in parasitic protozoa. Science. 228: 1443–1446, 1985

    PubMed  Google Scholar 

  22. Keegan F, Blum JJ: Effects of oxygen concentration on the intermediary metabolism ofLeishmania major promastigotes. Mol Biochem Parasit 39: 234–246, 1990

    Google Scholar 

  23. Rainey PM, Mackenzie NE: A carbon-13 nuclear magnetic resonance analysis of the products of glucose metabolism inLeishmania pifanoi amastigotes and promastigotes. Mol Biochem Parasit 45: 307–316, 1991

    Google Scholar 

  24. Darling TN, Davis DG, London RE, Blum JJ: Metabolism interactions between glucose, glycerol, alanine, and acetate inLeishmania braziliensis panamensis promastigotes. J Protoz 36: 217–225, 1989

    Google Scholar 

  25. Cazzulo JJ, Cazzulo BMFD, Engel JC, Cannata JJB: End products and enzyme levels of aerobic glucose fermentation in trypanosomatids. Mol Biochem Parasit 16: 329–343, 1985

    Google Scholar 

  26. Hart DT, Coombs GH:Leishmania mexicana: energy metabolism of amastigotes and promastigotes. Exp Parasit 54: 397–309, 1982

    PubMed  Google Scholar 

  27. Darling TN, Balber AE, Blum JJ: A comparative study of D-lactate, L-lactate and glycerol formation by four species ofLeishmania and byTrypanosoma lewisi andTrypanosoma brucei gambiense. Mol Biochem Parasit 30: 253–258, 1988

    Google Scholar 

  28. Simon MW, Martin E, Mukkada AJ: Evidence for a functional glyoxylate cycle in theLeishmaniae. J. Bacteriol 135: 895–899, 1978

    PubMed  Google Scholar 

  29. Mukkada AJ: Energy metabolism inLeishmania. In: KP Chang, RS Bray (eds),Leishmaniasis, Elsevier, Amsterdam, pp 49–64, 1985

    Google Scholar 

  30. Hart DT, Opperdoes FR: The occurrence of glycosomes (microbodies) in the promastigote stage of four majorLeishmania species. Mol Biochem Parasit 13: 159–172, 1984

    Google Scholar 

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Authors and Affiliations

  1. Instituto de Biotecnologia, Grupo de Investigación en Bioquímica y Parasitología Molecular, Facultad de Ciencias, Universidad de Granada, Campus Universitario Fuentenueva, 18071, Granada, Spain

    J. J. Castilla, M. Sanchez-Moreno & A. Osuna

  2. Departamento de Biologia Aplicada, Escuela Politécnica, Universidad de Almería, Almería, Spain

    C. Mesa

Authors
  1. J. J. Castilla
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  2. M. Sanchez-Moreno
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  3. C. Mesa
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  4. A. Osuna
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Castilla, J.J., Sanchez-Moreno, M., Mesa, C. et al. Leishmania donovani: In vitro culture and [1H] NMR characterization of amastigote-like forms. Mol Cell Biochem 142, 89–97 (1995). https://doi.org/10.1007/BF00928929

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  • DOI: https://doi.org/10.1007/BF00928929

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