Skip to main content
PMC home page
Antimicrobial Agents and Chemotherapy logoLink to Antimicrobial Agents and Chemotherapy
. 1996 Dec;40(12):2785–2791. doi: 10.1128/aac.40.12.2785

Naturally azole-resistant Leishmania braziliensis promastigotes are rendered susceptible in the presence of terbinafine: comparative study with azole-susceptible Leishmania mexicana promastigotes.

H Rangel 1, F Dagger 1, A Hernandez 1, A Liendo 1, J A Urbina 1
PMCID: PMC163622  PMID: 9124841

Abstract

Leishmania braziliensis (isolate 2903) was naturally resistant to ketoconazole or the bis-triazole D0870, inhibitors of sterol C-14 demethylase, which produced only moderate effects on the proliferation of promastigotes at 10 microM. In contrast, Leishmania mexicana (isolate NR) was extremely susceptible to the azoles, as complete growth arrest and cell lysis were induced by incubation of the parasites with 0.05 microM concentrations of the drugs for 72 h. The opposite response was observed with terbinafine, an inhibitor of squalene epoxidase: L. braziliensis 2903 was three times more susceptible to the drug than L. mexicana NR (MICs of 5 and 15 microM, respectively). However, when the L. braziliensis stock was grown in the presence of 1 microM terbinafine, which by itself produced only marginal (< 10%) effects on growth, it became highly susceptible to the azoles, with an MIC of 0.03 microM. Analysis of cellular free sterols by high-resolution capillary gas chromatography coupled to mass spectrometry showed that 14-methyl sterols can support normal growth of L. braziliensis 2903 but not of L. mexicana NR. On the other hand, the higher susceptibility of the L. braziliensis isolate to terbinafine was correlated with a massive accumulation of squalene in the presence of the allylamine while no significant effects on L. mexicana sterol composition were observed at drug concentrations up to 1 microM. Thus, the > 300-fold increase in the susceptibility of L. braziliensis promastigotes to azoles in the presence of terbinafine was attributed to the combined effect of squalene and the methylated sterol precursors on the physical properties of the cell's membranes, leading to the loss of cell viability. Combination therapy with azoles and terbinafine in the treatment of human L. braziliensis infections deserves further study.

Full Text

The Full Text of this article is available as a PDF (636.0 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Akuffo H., Dietz M., Teklemariam S., Tadesse T., Amare G., Berhan T. Y. The use of itraconazole in the treatment of leishmaniasis caused by Leishmania aethiopica. Trans R Soc Trop Med Hyg. 1990 Jul-Aug;84(4):532–534. doi: 10.1016/0035-9203(90)90028-d. [DOI] [PubMed] [Google Scholar]
  2. Arthington B. A., Bennett L. G., Skatrud P. L., Guynn C. J., Barbuch R. J., Ulbright C. E., Bard M. Cloning, disruption and sequence of the gene encoding yeast C-5 sterol desaturase. Gene. 1991 Jun 15;102(1):39–44. doi: 10.1016/0378-1119(91)90535-j. [DOI] [PubMed] [Google Scholar]
  3. Ashman W. H., Barbuch R. J., Ulbright C. E., Jarrett H. W., Bard M. Cloning and disruption of the yeast C-8 sterol isomerase gene. Lipids. 1991 Aug;26(8):628–632. doi: 10.1007/BF02536427. [DOI] [PubMed] [Google Scholar]
  4. Bard M., Lees N. D., Turi T., Craft D., Cofrin L., Barbuch R., Koegel C., Loper J. C. Sterol synthesis and viability of erg11 (cytochrome P450 lanosterol demethylase) mutations in Saccharomyces cerevisiae and Candida albicans. Lipids. 1993 Nov;28(11):963–967. doi: 10.1007/BF02537115. [DOI] [PubMed] [Google Scholar]
  5. Beach D. H., Goad L. J., Holz G. G., Jr Effects of antimycotic azoles on growth and sterol biosynthesis of Leishmania promastigotes. Mol Biochem Parasitol. 1988 Nov;31(2):149–162. doi: 10.1016/0166-6851(88)90166-1. [DOI] [PubMed] [Google Scholar]
  6. Berman J. D., Goad L. J., Beach D. H., Holz G. G., Jr Effects of ketoconazole on sterol biosynthesis by Leishmania mexicana mexicana amastigotes in murine macrophage tumor cells. Mol Biochem Parasitol. 1986 Jul;20(1):85–92. doi: 10.1016/0166-6851(86)90145-3. [DOI] [PubMed] [Google Scholar]
  7. Berman J. D., Holz G. G., Jr, Beach D. H. Effects of ketoconazole on growth and sterol biosynthesis of Leishmania mexicana promastigotes in culture. Mol Biochem Parasitol. 1984 May;12(1):1–13. doi: 10.1016/0166-6851(84)90039-2. [DOI] [PubMed] [Google Scholar]
  8. Bloch K. E. Sterol structure and membrane function. CRC Crit Rev Biochem. 1983;14(1):47–92. doi: 10.3109/10409238309102790. [DOI] [PubMed] [Google Scholar]
  9. Borelli D. A clinical trial of itraconazole in the treatment of deep mycoses and leishmaniasis. Rev Infect Dis. 1987 Jan-Feb;9 (Suppl 1):S57–S63. doi: 10.1093/clinids/9.supplement_1.s57. [DOI] [PubMed] [Google Scholar]
  10. Buttke T. M., Bloch K. Utilization and metabolism of methyl-sterol derivatives in the yeast mutant strain GL7. Biochemistry. 1981 May 26;20(11):3267–3272. doi: 10.1021/bi00514a044. [DOI] [PubMed] [Google Scholar]
  11. Croft S. L. Recent developments in the chemotherapy of leishmaniasis. Trends Pharmacol Sci. 1988 Oct;9(10):376–381. doi: 10.1016/0165-6147(88)90258-1. [DOI] [PubMed] [Google Scholar]
  12. Dahl J. S., Dahl C. E., Bloch K. Sterols in membranes: growth characteristics and membrane properties of Mycoplasma capricolum cultured on cholesterol and lanosterol. Biochemistry. 1980 Apr 1;19(7):1467–1472. doi: 10.1021/bi00548a032. [DOI] [PubMed] [Google Scholar]
  13. Dan M., Verner E., el-On J., Zuckerman F., Michaeli D. Failure of oral ketoconazole to cure cutaneous ulcers caused by Leishmania braziliensis. Cutis. 1986 Sep;38(3):198–199. [PubMed] [Google Scholar]
  14. Ellenberger T. E., Beverley S. M. Multiple drug resistance and conservative amplification of the H region in Leishmania major. J Biol Chem. 1989 Sep 5;264(25):15094–15103. [PubMed] [Google Scholar]
  15. Gaber R. F., Copple D. M., Kennedy B. K., Vidal M., Bard M. The yeast gene ERG6 is required for normal membrane function but is not essential for biosynthesis of the cell-cycle-sparking sterol. Mol Cell Biol. 1989 Aug;9(8):3447–3456. doi: 10.1128/mcb.9.8.3447. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Goad L. J., Holz G. G., Jr, Beach D. H. Sterols of Leishmania species. Implications for biosynthesis. Mol Biochem Parasitol. 1984 Feb;10(2):161–170. doi: 10.1016/0166-6851(84)90004-5. [DOI] [PubMed] [Google Scholar]
  17. Goad L. J., Holz G. G., Jr, Beach D. H. Sterols of ketoconazole-inhibited Leishmania mexicana mexicana promastigotes. Mol Biochem Parasitol. 1985 Jun;15(3):257–279. doi: 10.1016/0166-6851(85)90089-1. [DOI] [PubMed] [Google Scholar]
  18. Hart D. T., Lauwers W. J., Willemsens G., Vanden Bossche H., Opperdoes F. R. Perturbation of sterol biosynthesis by itraconazole and ketoconazole in Leishmania mexicana mexicana infected macrophages. Mol Biochem Parasitol. 1989 Mar 1;33(2):123–134. doi: 10.1016/0166-6851(89)90026-1. [DOI] [PubMed] [Google Scholar]
  19. Haughan P. A., Chance M. L., Goad L. J. Effects of an azasterol inhibitor of sterol 24-transmethylation on sterol biosynthesis and growth of Leishmania donovani promastigotes. Biochem J. 1995 May 15;308(Pt 1):31–38. doi: 10.1042/bj3080031. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Haughan P. A., Chance M. L., Goad L. J. Effects of sinefungin on growth and sterol composition of Leishmania promastigotes. Exp Parasitol. 1993 Sep;77(2):147–154. doi: 10.1006/expr.1993.1071. [DOI] [PubMed] [Google Scholar]
  21. Haughan P. A., Chance M. L., Goad L. J. Synergism in vitro of lovastatin and miconazole as anti-leishmanial agents. Biochem Pharmacol. 1992 Dec 1;44(11):2199–2206. doi: 10.1016/0006-2952(92)90347-l. [DOI] [PubMed] [Google Scholar]
  22. Hernandez A. G., Rascon A., Kutner S., Roman H., Campos Z. Relationships between cell surface protease and acid phosphatase activities of Leishmania promastigote. Mol Biol Rep. 1993 Oct;18(3):189–195. doi: 10.1007/BF01674430. [DOI] [PubMed] [Google Scholar]
  23. Herwaldt B. L., Berman J. D. Recommendations for treating leishmaniasis with sodium stibogluconate (Pentostam) and review of pertinent clinical studies. Am J Trop Med Hyg. 1992 Mar;46(3):296–306. doi: 10.4269/ajtmh.1992.46.296. [DOI] [PubMed] [Google Scholar]
  24. Huang T. H., DeSiervo A. J., Yang Q. X. Effect of cholesterol and lanosterol on the structure and dynamics of the cell membrane of Mycoplasma capricolum. Deuterium nuclear magnetic resonance study. Biophys J. 1991 Mar;59(3):691–702. doi: 10.1016/S0006-3495(91)82283-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Jolliffe D. S. Cutaneous leishmaniasis from Belize--treatment with ketoconazole. Clin Exp Dermatol. 1986 Jan;11(1):62–68. doi: 10.1111/j.1365-2230.1986.tb00425.x. [DOI] [PubMed] [Google Scholar]
  26. Larralde G., Vivas J., Urbina J. A. Concentration and time dependence of the effects of ketoconazole on growth and sterol synthesis by Trypanosoma (Schizotrypanum) cruzi epimastigotes. Acta Cient Venez. 1988;39(2):140–146. [PubMed] [Google Scholar]
  27. Maldonado R. A., Molina J., Payares G., Urbina J. A. Experimental chemotherapy with combinations of ergosterol biosynthesis inhibitors in murine models of Chagas' disease. Antimicrob Agents Chemother. 1993 Jun;37(6):1353–1359. doi: 10.1128/aac.37.6.1353. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Navin T. R., Arana B. A., Arana F. E., Berman J. D., Chajón J. F. Placebo-controlled clinical trial of sodium stibogluconate (Pentostam) versus ketoconazole for treating cutaneous leishmaniasis in Guatemala. J Infect Dis. 1992 Mar;165(3):528–534. doi: 10.1093/infdis/165.3.528. [DOI] [PubMed] [Google Scholar]
  29. Nes W. D., Janssen G. G., Crumley F. G., Kalinowska M., Akihisa T. The structural requirements of sterols for membrane function in Saccharomyces cerevisiae. Arch Biochem Biophys. 1993 Feb 1;300(2):724–733. doi: 10.1006/abbi.1993.1100. [DOI] [PubMed] [Google Scholar]
  30. Norton S. A., Frankenburg S., Klaus S. N. Cutaneous leishmaniasis acquired during military service in the Middle East. Arch Dermatol. 1992 Jan;128(1):83–87. [PubMed] [Google Scholar]
  31. Olliaro P. L., Bryceson A. D. Practical progress and new drugs for changing patterns of leishmaniasis. Parasitol Today. 1993 Sep;9(9):323–328. doi: 10.1016/0169-4758(93)90231-4. [DOI] [PubMed] [Google Scholar]
  32. Parks L. W., Casey W. M. Physiological implications of sterol biosynthesis in yeast. Annu Rev Microbiol. 1995;49:95–116. doi: 10.1146/annurev.mi.49.100195.000523. [DOI] [PubMed] [Google Scholar]
  33. Ramírez J. L., Guevara P. The ribosomal gene spacer as a tool for the taxonomy of Leishmania. Mol Biochem Parasitol. 1987 Jan 15;22(2-3):177–183. doi: 10.1016/0166-6851(87)90048-x. [DOI] [PubMed] [Google Scholar]
  34. Ryder N. S., Mieth H. Allylamine antifungal drugs. Curr Top Med Mycol. 1992;4:158–188. doi: 10.1007/978-1-4612-2762-5_6. [DOI] [PubMed] [Google Scholar]
  35. Saenz R. E., Paz H., Berman J. D. Efficacy of ketoconazole against Leishmania braziliensis panamensis cutaneous leishmaniasis. Am J Med. 1990 Aug;89(2):147–155. doi: 10.1016/0002-9343(90)90292-l. [DOI] [PubMed] [Google Scholar]
  36. Urbina J. A., Lazardi K., Aguirre T., Piras M. M., Piras R. Antiproliferative effects and mechanism of action of ICI 195,739, a novel bis-triazole derivative, on epimastigotes and amastigotes of Trypanosoma (Schizotrypanum) cruzi. Antimicrob Agents Chemother. 1991 Apr;35(4):730–735. doi: 10.1128/aac.35.4.730. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Urbina J. A., Lazardi K., Aguirre T., Piras M. M., Piras R. Antiproliferative synergism of the allylamine SF 86-327 and ketoconazole on epimastigotes and amastigotes of Trypanosoma (Schizotrypanum) cruzi. Antimicrob Agents Chemother. 1988 Aug;32(8):1237–1242. doi: 10.1128/aac.32.8.1237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Urbina J. A., Lazardi K., Marchan E., Visbal G., Aguirre T., Piras M. M., Piras R., Maldonado R. A., Payares G., de Souza W. Mevinolin (lovastatin) potentiates the antiproliferative effects of ketoconazole and terbinafine against Trypanosoma (Schizotrypanum) cruzi: in vitro and in vivo studies. Antimicrob Agents Chemother. 1993 Mar;37(3):580–591. doi: 10.1128/aac.37.3.580. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Urbina J. A., Pekerar S., Le H. B., Patterson J., Montez B., Oldfield E. Molecular order and dynamics of phosphatidylcholine bilayer membranes in the presence of cholesterol, ergosterol and lanosterol: a comparative study using 2H-, 13C- and 31P-NMR spectroscopy. Biochim Biophys Acta. 1995 Sep 13;1238(2):163–176. doi: 10.1016/0005-2736(95)00117-l. [DOI] [PubMed] [Google Scholar]
  40. Urbina J. A., Vivas J., Lazardi K., Molina J., Payares G., Piras M. M., Piras R. Antiproliferative effects of delta 24(25) sterol methyl transferase inhibitors on Trypanosoma (Schizotrypanum) cruzi: in vitro and in vivo studies. Chemotherapy. 1996 Jul-Aug;42(4):294–307. doi: 10.1159/000239458. [DOI] [PubMed] [Google Scholar]
  41. Urbina J. A., Vivas J., Visbal G., Contreras L. M. Modification of the sterol composition of Trypanosoma (Schizotrypanum) cruzi epimastigotes by delta 24(25)-sterol methyl transferase inhibitors and their combinations with ketoconazole. Mol Biochem Parasitol. 1995 Jul;73(1-2):199–210. doi: 10.1016/0166-6851(95)00117-j. [DOI] [PubMed] [Google Scholar]
  42. Urcuyo F. G., Zaias N. Oral ketoconazole in the treatment of leishmaniasis. Int J Dermatol. 1982 Sep;21(7):414–416. doi: 10.1111/j.1365-4362.1982.tb03163.x. [DOI] [PubMed] [Google Scholar]
  43. Vanden Bossche H., Marichal P., Gorrens J., Geerts H., Janssen P. A. Mode of action studies. Basis for the search of new antifungal drugs. Ann N Y Acad Sci. 1988;544:191–207. doi: 10.1111/j.1749-6632.1988.tb40404.x. [DOI] [PubMed] [Google Scholar]
  44. Vannier-Santos M. A., Urbina J. A., Martiny A., Neves A., de Souza W. Alterations induced by the antifungal compounds ketoconazole and terbinafine in Leishmania. J Eukaryot Microbiol. 1995 Jul-Aug;42(4):337–346. doi: 10.1111/j.1550-7408.1995.tb01591.x. [DOI] [PubMed] [Google Scholar]
  45. Weinrauch L., Livshin R., Even-Paz Z., El-On J. Efficacy of ketoconazole in cutaneous leishmaniasis. Arch Dermatol Res. 1983;275(5):353–354. doi: 10.1007/BF00417211. [DOI] [PubMed] [Google Scholar]
  46. Weinrauch L., Livshin R., el-On J. Ketoconazole in cutaneous leishmaniasis. Br J Dermatol. 1987 Nov;117(5):666–668. doi: 10.1111/j.1365-2133.1987.tb07504.x. [DOI] [PubMed] [Google Scholar]
  47. Yeagle P. L. Lanosterol and cholesterol have different effects on phospholipid acyl chain ordering. Biochim Biophys Acta. 1985 Apr 26;815(1):33–36. doi: 10.1016/0005-2736(85)90470-5. [DOI] [PubMed] [Google Scholar]
  48. de Maio A., Urbina J. A. Trypanosoma (Schizotrypanum) cruzi: terminal oxidases in exponential and stationary growth phase emipastigotes cultured in vitro. Acta Cient Venez. 1984;35(2):136–141. [PubMed] [Google Scholar]

Articles from Antimicrobial Agents and Chemotherapy are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES