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. 1997 Sep;41(9):1898–1903. doi: 10.1128/aac.41.9.1898

In vitro life cycle of pentamidine-resistant amastigotes: stability of the chemoresistant phenotypes is dependent on the level of resistance induced.

D Sereno 1, J L Lemesre 1
PMCID: PMC164032  PMID: 9303381

Abstract

Using a continuous drug pressure protocol, we induced pentamidine resistance in an active and dividing population of amastigote forms of Leishmania mexicana. We selected in vitro two clones with different levels of resistance to pentamidine, with clone LmPENT5 being resistant to 5 microM pentamidine, while clone LmPENT20 was resistant to 20 microM pentamidine. Resistance indexes (50% inhibitory concentration [IC50] after drug presure/IC50 before drug pressure) of 2 (LmPENT5) and 6 (LmPENT20) were determined after drug selection. Both resistant clones expressed significant cross-resistance to diminazene aceturate and primaquine. Pentamidine resistance was not reversed by verapamil, a calcium channel blocker known to reverse multidrug resistance (A. J. Bitonti, et al., Science 242:1301-1303, 1988; A. R. C. Safa et al., J. Biol. Chem. 262:7884-7888, 1987). No difference in the in vitro infectivity for resident mouse macrophages was observed between the wild-type clone (clone LmWT) and pentamidine-resistant clones. During in vitro infectivity experiments, when the life cycle was performed starting from the intramacrophagic amastigote stage, the drug resistance of the resulting LmPENT20 amastigotes was preserved even if the intermediate promastigote stage could not be considered resistant to 20 microM pentamidine. In the same way, when a complete developmental sequence of L. mexicana was achieved axenically by manipulation of appropriate culture conditions, the resulting axenically grown LmPENT20 amastigotes remained pentamidine resistant, whereas LmPENT5 amastigotes lost their ability to resist pentamidine, with IC50s and index of resistance values close to those for the LmWT clone. These results strongly indicate that the level of pentamidine tolerated by resistant amastigotes after the life cycle was dependent on the induced level of resistance. This fact could be significant in the in vivo transmission of drug-resistant parasites by Phlebotominae. Particular attention should be given to the finding that the emergence of parasite resistance is a potential risk of the use of inadequate doses as therapy in humans.

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Selected References

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  1. Basselin M., Lawrence F., Robert-Gero M. Pentamidine uptake in Leishmania donovani and Leishmania amazonensis promastigotes and axenic amastigotes. Biochem J. 1996 Apr 15;315(Pt 2):631–634. doi: 10.1042/bj3150631. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bates P. A. Axenic culture of Leishmania amastigotes. Parasitol Today. 1993 Apr;9(4):143–146. doi: 10.1016/0169-4758(93)90181-e. [DOI] [PubMed] [Google Scholar]
  3. Bates P. A. Complete developmental cycle of Leishmania mexicana in axenic culture. Parasitology. 1994 Jan;108(Pt 1):1–9. doi: 10.1017/s0031182000078458. [DOI] [PubMed] [Google Scholar]
  4. Bates P. A., Robertson C. D., Tetley L., Coombs G. H. Axenic cultivation and characterization of Leishmania mexicana amastigote-like forms. Parasitology. 1992 Oct;105(Pt 2):193–202. doi: 10.1017/s0031182000074102. [DOI] [PubMed] [Google Scholar]
  5. Berman J. D., Gallalee J. V., Hansen B. D. Leishmania mexicana: uptake of sodium stibogluconate (Pentostam) and pentamidine by parasite and macrophages. Exp Parasitol. 1987 Aug;64(1):127–131. doi: 10.1016/0014-4894(87)90018-x. [DOI] [PubMed] [Google Scholar]
  6. Bitonti A. J., Sjoerdsma A., McCann P. P., Kyle D. E., Oduola A. M., Rossan R. N., Milhous W. K., Davidson D. E., Jr Reversal of chloroquine resistance in malaria parasite Plasmodium falciparum by desipramine. Science. 1988 Dec 2;242(4883):1301–1303. doi: 10.1126/science.3057629. [DOI] [PubMed] [Google Scholar]
  7. Bronner U., Doua F., Ericsson O., Gustafsson L. L., Miézan T. W., Rais M., Rombo L. Pentamidine concentrations in plasma, whole blood and cerebrospinal fluid during treatment of Trypanosoma gambiense infection in Côte d'Ivoire. Trans R Soc Trop Med Hyg. 1991 Sep-Oct;85(5):608–611. doi: 10.1016/0035-9203(91)90364-5. [DOI] [PubMed] [Google Scholar]
  8. Coombs G. H., Craft J. A., Hart D. T. A comparative study of Leishmania mexicana amastigotes and promastigotes. Enzyme activities and subcellular locations. Mol Biochem Parasitol. 1982 Mar;5(3):199–211. doi: 10.1016/0166-6851(82)90021-4. [DOI] [PubMed] [Google Scholar]
  9. Coombs G. H., Tetley L., Moss V. A., Vickerman K. Three dimensional structure of the Leishmania amastigote as revealed by computer-aided reconstruction from serial sections. Parasitology. 1986 Feb;92(Pt 1):13–23. doi: 10.1017/s0031182000063411. [DOI] [PubMed] [Google Scholar]
  10. Coons T., Hanson S., Bitonti A. J., McCann P. P., Ullman B. Alpha-difluoromethylornithine resistance in Leishmania donovani is associated with increased ornithine decarboxylase activity. Mol Biochem Parasitol. 1990 Feb;39(1):77–89. doi: 10.1016/0166-6851(90)90010-j. [DOI] [PubMed] [Google Scholar]
  11. Endicott J. A., Ling V. The biochemistry of P-glycoprotein-mediated multidrug resistance. Annu Rev Biochem. 1989;58:137–171. doi: 10.1146/annurev.bi.58.070189.001033. [DOI] [PubMed] [Google Scholar]
  12. Flintoff W. F., Essani K. Methotrexate-resistant Chinese hamster ovary cells contain a dihydrofolate reductase with an altered affinity for methotrexate. Biochemistry. 1980 Sep 2;19(18):4321–4327. doi: 10.1021/bi00559a027. [DOI] [PubMed] [Google Scholar]
  13. Grogl M., Thomason T. N., Franke E. D. Drug resistance in leishmaniasis: its implication in systemic chemotherapy of cutaneous and mucocutaneous disease. Am J Trop Med Hyg. 1992 Jul;47(1):117–126. doi: 10.4269/ajtmh.1992.47.117. [DOI] [PubMed] [Google Scholar]
  14. Jackson J. E., Tally J. D., Ellis W. Y., Mebrahtu Y. B., Lawyer P. G., Were J. B., Reed S. G., Panisko D. M., Limmer B. L. Quantitative in vitro drug potency and drug susceptibility evaluation of Leishmania ssp. from patients unresponsive to pentavalent antimony therapy. Am J Trop Med Hyg. 1990 Nov;43(5):464–480. doi: 10.4269/ajtmh.1990.43.464. [DOI] [PubMed] [Google Scholar]
  15. Jackson J. E., Tally J. D., Tang D. B. An in vitro micromethod for drug sensitivity testing of Leishmania. Am J Trop Med Hyg. 1989 Sep;41(3):318–330. [PubMed] [Google Scholar]
  16. Kweider M., Lemesre J. L., Santoro F., Kusnierz J. P., Sadigursky M., Capron A. Development of metacyclic Leishmania promastigotes is associated with the increasing expression of GP65, the major surface antigen. Parasite Immunol. 1989 May;11(3):197–209. doi: 10.1111/j.1365-3024.1989.tb00659.x. [DOI] [PubMed] [Google Scholar]
  17. Lemesre J. L., Sereno D., Daulouède S., Veyret B., Brajon N., Vincendeau P. Leishmania spp.: nitric oxide-mediated metabolic inhibition of promastigote and axenically grown amastigote forms. Exp Parasitol. 1997 May;86(1):58–68. doi: 10.1006/expr.1997.4151. [DOI] [PubMed] [Google Scholar]
  18. Ouellette M., Papadopoulou B. Mechanisms of drug resistance in Leishmania. Parasitol Today. 1993 May;9(5):150–153. doi: 10.1016/0169-4758(93)90135-3. [DOI] [PubMed] [Google Scholar]
  19. Pan A. A., Pan S. C. Leishmania mexicana: comparative fine structure of amastigotes and promastigotes in vitro and in vivo. Exp Parasitol. 1986 Oct;62(2):254–265. doi: 10.1016/0014-4894(86)90030-5. [DOI] [PubMed] [Google Scholar]
  20. Peters B. S., Fish D., Golden R., Evans D. A., Bryceson A. D., Pinching A. J. Visceral leishmaniasis in HIV infection and AIDS: clinical features and response to therapy. Q J Med. 1990 Nov;77(283):1101–1111. doi: 10.1093/qjmed/77.2.1101. [DOI] [PubMed] [Google Scholar]
  21. Rainey P. M., Spithill T. W., McMahon-Pratt D., Pan A. A. Biochemical and molecular characterization of Leishmania pifanoi amastigotes in continuous axenic culture. Mol Biochem Parasitol. 1991 Nov;49(1):111–118. doi: 10.1016/0166-6851(91)90134-r. [DOI] [PubMed] [Google Scholar]
  22. Safa A. R., Glover C. J., Sewell J. L., Meyers M. B., Biedler J. L., Felsted R. L. Identification of the multidrug resistance-related membrane glycoprotein as an acceptor for calcium channel blockers. J Biol Chem. 1987 Jun 5;262(16):7884–7888. [PubMed] [Google Scholar]
  23. Sereno D., Lemesre J. L. Axenically cultured amastigote forms as an in vitro model for investigation of antileishmanial agents. Antimicrob Agents Chemother. 1997 May;41(5):972–976. doi: 10.1128/aac.41.5.972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Sereno D., Lemesre J. L. Use of an enzymatic micromethod to quantify amastigote stage of Leishmania amazonensis in vitro. Parasitol Res. 1997;83(4):401–403. doi: 10.1007/s004360050272. [DOI] [PubMed] [Google Scholar]
  25. Sundar S., Thakur B. B., Tandon A. K., Agrawal N. R., Mishra C. P., Mahapatra T. M., Singh V. P. Clinicoepidemiological study of drug resistance in Indian kala-azar. BMJ. 1994 Jan 29;308(6924):307–307. doi: 10.1136/bmj.308.6924.307. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Ullman B. Multidrug resistance and P-glycoproteins in parasitic protozoa. J Bioenerg Biomembr. 1995 Feb;27(1):77–84. doi: 10.1007/BF02110334. [DOI] [PubMed] [Google Scholar]
  27. Waalkes T. P., DeVita V. T. he determination of pentamidine (4,4'-diamidinophenoxypentane) in plasma, urine, and tissues. J Lab Clin Med. 1970 May;75(5):871–878. [PubMed] [Google Scholar]

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