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Tomatidine promotes the inhibition of 24-alkylated sterol biosynthesis and mitochondrial dysfunction in Leishmania amazonensis promastigotes

Published online by Cambridge University Press:  01 May 2012

J. M. MEDINA ,
J. C. F. RODRIGUES ,
W. DE SOUZA ,
G. C. ATELLA  and
H. BARRABIN
J. M. MEDINA
Affiliation:
Instituto de Bioquímica Médica, UFRJ, Rio de Janeiro, Brasil
J. C. F. RODRIGUES
Affiliation:
Instituto de Biofísica Carlos Chagas Filho, UFRJ, Rio de Janeiro, Brasil Pólo Avançado de Xerém, Universidade Federal do Rio de Janeiro, Brasil
W. DE SOUZA
Affiliation:
Instituto de Biofísica Carlos Chagas Filho, UFRJ, Rio de Janeiro, Brasil
G. C. ATELLA
Affiliation:
Instituto de Bioquímica Médica, UFRJ, Rio de Janeiro, Brasil
H. BARRABIN*
Affiliation:
Instituto de Bioquímica Médica, UFRJ, Rio de Janeiro, Brasil
*
*Corresponding author: Instituto de Bioquímica Médica, Programa de Biologia Estrutural – CCS, Universidade Federal do Rio de Janeiro-UFRJ, Ilha do Fundão, 21941-590 - Rio de Janeiro, Brazil. Tel: +55 21 2590 4548. Fax: +55 21 2562 6787. E-mail: [email protected]
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Summary

Leishmaniasis is a set of clinically distinct infectious diseases caused by Leishmania, a genus of flagellated protozoan parasites, that affects ∼12 million people worldwide, with ∼2 million new infections annually. Plants are known to produce substances to defend themselves against pathogens and predators. In the genus Lycopersicon, which includes the tomato, L. esculentum, the main antimicrobial compound is the steroidal glycoalkaloid α-tomatine. The loss of the saccharide side-chain of tomatine yields the aglycone tomatidine. In the present study, we investigated the effects of tomatidine on the growth, mitochondrial membrane potential, sterol metabolism, and ultrastructure of Leishmania amazonensis promastigotes. Tomatidine (0·1 to 5 μM) inhibited parasite growth in a dose-dependent manner (IC50=124±59 nM). Transmission electron microscopy revealed lesions in the mitochondrial ultrastructure and the presence of large vacuoles and lipid storage bodies in the cytoplasm. These structural changes in the mitochondria were accompanied by an effective loss of mitochondrial membrane potential and a decrease in ATP levels. An analysis of the neutral lipid content revealed a large depletion of endogenous 24-alkylated sterols such as 24-methylene-cholesta-5, 7-dien-3β-ol (5-dehydroepisterol), with a concomitant accumulation of cholesta-8, 24-dien-3β-ol (zymosterol), which implied a perturbation in the cellular lipid content. These results are consistent with an inhibition of 24-sterol methyltransferase, an important enzyme responsible for the methylation of sterols at the 24 position, which is an essential step in the production of ergosterol and other 24-methyl sterols.

Keywords

Leishmaniatomatidinesterols24-sterol methyltransferasemitochondriatrypanosomatids
Type
Research Article
Information
Parasitology , Volume 139 , Issue 10 , September 2012 , pp. 1253 - 1265
Copyright
Copyright © Cambridge University Press 2012

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References

REFERENCES

Blankemeyer, J. T., White, J. B., Stringer, B. K. and Friedman, M. (1997). Effect of alpha-tomatine and tomatidine on membrane potential of frog embryos and active transport of ions in frog skin. Food and Chemical Toxicology 35, 639–646. doi: 10.1016/S0278-6915(97)00038-0.CrossRefGoogle ScholarPubMed
Bligh, E. G. and Dyer, W. J. (1959). A rapid method of total lipid extraction and purification. Canadian Journal of Biochemistry and Physiology 37, 911–917. doi: 10.1139/o59-099.CrossRefGoogle ScholarPubMed
Cossarizza, A., Baccarani-Contri, M. G., Kalashnikova, and Franceschi, C. (1993). A new method for the cytofluorimetric analysis of mitochondrial membrane potential using the J-aggregate forming lipophilic cation 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanine iodide (JC-1). Biochemical and Biophysical Research Communications 197, 40–45. doi: 10.1006/bbrc.1993.2438.CrossRefGoogle Scholar
Croft, S. L., Barrett, M. P. and Urbina, J. A. (2005). Chemotherapy of trypanosomiases and leishmaniasis. Trends in Parasitology 21, 508–512. doi: 10.1016/j.pt.2005.08.026.CrossRefGoogle ScholarPubMed
Croft, S. L., Sundar, S. and Fairlamb, A. H. (2006). Drug resistance in leishmaniasis. Clinical Microbiology Reviews 19, 111–126. doi: 10.1128/CMR.19.1.111-126.2006.CrossRefGoogle ScholarPubMed
Cuba-Cuba, C. A., Miles, M. A., Vexenat, A., Barker, D. C., McMahon Pratt, D., Butcher, J., Barreto, A. C. and Marsden, P. D. (1985). A focus of mucocutaneous leishmaniasis in Tres Bracos, Bahia, Brazil: characterization and identification of Leishmania stocks isolated from man and dogs. Transactions of the Royal Society of Tropical Medicine and Hygiene 79, 500–507. doi: 10.1016/0035-9203(85)90077-X.CrossRefGoogle ScholarPubMed
de Souza, W. and Rodrigues, J. C. (2009). Sterol biosynthesis pathway as target for anti-trypanosomatid drugs. Interdisciplinary Perspectives on Infectious Diseases 2009, article ID, 1–19. 642502. doi: 10.1155/2009/642502.CrossRefGoogle ScholarPubMed
Desjeux, P. (1996). Leishmaniasis. Public health aspects and control. Clinical Dermatology 14, 417–423. doi: 10.1016/0738-081X(96)00057-0.CrossRefGoogle Scholar
Dixon, R. A. (2001). Natural products and plant disease resistance. Nature, London 411, 843–847. doi: 10.1038/35081178.CrossRefGoogle ScholarPubMed
Friedman, M. (2002). Tomato glycoalkaloids: role in the plant and in the diet. The Journal of Agricultural and Food Chemistry 50, 5751–5780. doi: 10.1021/jf020560c.CrossRefGoogle ScholarPubMed
Goad, L. J., Holz, G. G. Jr. and Beach, D. H. (1984). Sterols of Leishmania species. Implications for biosynthesis. Molecular and Biochemical Parasitology 10, 161–170. doi: 10.1016/0166-6851(84)90004-5.CrossRefGoogle ScholarPubMed
Granthon, A. C., Braga, M. V., Rodrigues, J. C., Cammerer, S., Lorente, S. O., Gilbert, I. H., Urbina, J. A. and de Souza, W. (2007). Alterations on the growth and ultrastructure of Leishmania chagasi induced by squalene synthase inhibitors. Veterinary Parasitology 146, 25–34. doi: 10.1016/j.vetpar.2006.12.022.CrossRefGoogle ScholarPubMed
Greenspan, P., Mayer, E. P. and Fowler, S. D. (1985). Nile red: a selective fluorescent stain for intracellular lipid droplets. The Journal of Cell Biology 100, 965–973.CrossRefGoogle ScholarPubMed
Hankins, E. G., Gillespie, J. R., Aikenhead, K. and Buckner, F. S. (2005). Upregulation of sterol C14-demethylase expression in Trypanosoma cruzi treated with sterol biosynthesis inhibitors. Molecular and Biochemical Parasitology 144, 68–75. doi: 10.1016/j.molbiopara.2005.08.002.CrossRefGoogle ScholarPubMed
Haughan, P. A., Chance, M. L. and Goad, L. J. (1995). Effects of an azasterol inhibitor of sterol 24-transmethylation on sterol biosynthesis and growth of Leishmania donovani promastigotes. The Biochemical Journal 308, 31–38.CrossRefGoogle ScholarPubMed
Johnson, L. V., Walsh, M. L., Bockus, B. J. and Chen, L. B. (1981). Monitoring of relative mitochondrial membrane potential in living cells by fluorescence microscopy. The Journal of Cell Biology 88, 527–535.CrossRefGoogle ScholarPubMed
Lazardi, K., Urbina, J. A. and de Souza, W. (1990). Ultrastructural alterations induced by two ergosterol biosynthesis inhibitors, ketoconazole and terbinafine, on epimastigotes and amastigotes of Trypanosoma (Schizotrypanum) cruzi . Antimicrobial Agents and Chemotherapy 34, 2097–2105.CrossRefGoogle ScholarPubMed
Lazardi, K., Urbina, J. A. and de Souza, W. (1991). Ultrastructural alterations induced by ICI 195,739, a bis-triazole derivative with strong antiproliferative action against Trypanosoma (Schizotrypanum) cruzi . Antimicrobial Agents and Chemotherapy 35, 736–740.CrossRefGoogle ScholarPubMed
Lorente, S. O., Rodrigues, J. C., Jiménez Jiménez, C., Joyce-Menekse, M., Rodrigues, C., Croft, S. L., Yardley, V., de Luca-Fradley, K., Ruiz-Pérez, L. M., Urbina, J., de Souza, W., González Pacanowska, D. and Gilbert, I. H. (2004). Novel azasterols as potential agents for treatment of leishmaniasis and trypanosomiasis. Antimicrobial Agents and Chemotherapy 48, 2937–2950. doi: 10.1128/AAC.48.8.2937–2950.2004 CrossRefGoogle ScholarPubMed
Mangla, A. T. and Nes, W. D. (2000). Sterol C-methyl transferase from Prototheca wickerhamii mechanism, sterol specificity and inhibition. Bioorganic & Medicinal Chemistry 8, 925–936. doi: 10.1016/S0968-0896(00)00040-7.CrossRefGoogle ScholarPubMed
Mehta, A. and Shaha, C. (2004). Apoptotic death in Leishmania donovani promastigotes in response to respiratory chain inhibition: complex II inhibition results in increased pentamidine cytotoxicity. The Journal of Biological Chemistry 279, 11798–11813. doi: 10.1074/jbc.M309341200.CrossRefGoogle ScholarPubMed
Mukherjee, S. B., Das, M., Sudhandiran, G. and Shaha, C. (2002). Increase in cytosolic Ca2+ levels through the activation of non-selective cation channels induced by oxidative stress causes mitochondrial depolarization leading to apoptosis-like death in Leishmania donovani promastigotes. The Journal of Biological Chemistry 277, 24717–24727. doi: 10.1074/jbc.M201961200.CrossRefGoogle ScholarPubMed
Nes, W. D. (2000). Sterol methyl transferase: enzymology and inhibition. Biochimica et Biophysica Acta 1529, 63–88. doi: 10.1016/S1388-1981(00)00138-4.CrossRefGoogle ScholarPubMed
Palmie-Peixoto, I. V., Rocha, M. R., Urbina, J. A., de Souza, W., Einicker-Lamas, M. and Motta, M. C. (2006). Effects of sterol biosynthesis inhibitors on endosymbiont-bearing trypanosomatids. FEMS Microbiology Letters 255, 33–42. doi: 10.1111/j.1574-6968.2005.00056.x.CrossRefGoogle ScholarPubMed
Pichler, H. and Riezman, H. (2004). Where sterols are required for endocytosis. Biochimica et Biophysica Acta 1666, 51–61. doi: 10.1016/j.bbamem.2004.05.011.CrossRefGoogle ScholarPubMed
Roberts, C. W., McLeod, R., Rice, D. W., Ginger, M., Chance, M. L. and Goad, L. J. (2003). Fatty acid and sterol metabolism: potential antimicrobial targets in apicomplexan and trypanosomatid parasitic protozoa. Molecular and Biochemical Parasitology 126, 129–142. doi: 10.1016/S0166-6851(02)00280-3.CrossRefGoogle ScholarPubMed
Rodrigues, C. O., Catisti, R., Uyemura, S. A., Vercesi, A. E., Lira, R., Rodriguez, C., Urbina, J. A. and Docampo, R. (2001). The sterol composition of Trypanosoma cruzi changes after growth in different culture media and results in different sensitivity to digitonin-permeabilization. Journal of Eukaryotic Microbiology 48, 588–594. doi: 10.1111/j.1550-7408.2001.tb00195.x.CrossRefGoogle ScholarPubMed
Rodrigues, J. C., Attias, M., Rodriguez, C., Urbina, J. A. and Souza, W. (2002). Ultrastructural and biochemical alterations induced by 22,26-azasterol, a delta(24(25))-sterol methyltransferase inhibitor, on promastigote and amastigote forms of Leishmania amazonensis . Antimicrobial Agents and Chemotherapy 46, 487–499. doi: 10.1128/AAC.46.2.487-499.2002.CrossRefGoogle Scholar
Rodrigues, J. C., Bernardes, C. F., Visbal, G., Urbina, J. A., Vercesi, A. E. and de Souza, W. (2007). Sterol methenyl transferase inhibitors alter the ultrastructure and function of the Leishmania amazonensis mitochondrion leading to potent growth inhibition. Protist 158, 447–456. doi: 10.1016/j.protis.2007.05.004.CrossRefGoogle ScholarPubMed
Rodrigues, J. C. and de Souza, W. (2008). Ultrastructural alterations in organelles of parasitic protozoa induced by different classes of metabolic inhibitors. Current Pharmaceutical Design 14, 925–938.CrossRefGoogle ScholarPubMed
Rodrigues, J. C., Urbina, J. A. and de Souza, W. (2005). Antiproliferative and ultrastructural effects of BPQ-OH, a specific inhibitor of squalene synthase, on Leishmania amazonensis . Experimental Parasitology 111, 230–238. doi: 10.1016/j.exppara.2005.08.006.CrossRefGoogle ScholarPubMed
Santa-Rita, R. M., Lira, R., Barbosa, H. S., Urbina, J. A. and de Castro, S. L. (2005). Anti-proliferative synergy of lysophospholipid analogues and ketoconazole against Trypanosoma cruzi (Kinetoplastida: Trypanosomatidae): cellular and ultrastructural analysis. The Journal of Antimicrobial Chemotherapy 55, 780–784. doi: 10.1093/jac/dki087 CrossRefGoogle ScholarPubMed
Saudagar, P. and Dubey, V. K. (2011). Cloning, expression, characterization and inhibition studies on trypanothione synthetase, a drug target enzyme, from Leishmania donovani. Biological Chemistry 392, 1113–1122. doi: 10.1515/BC.2011.222.CrossRefGoogle ScholarPubMed
Sen, N., Banerjee, B., Gupta, S. S., Das, B. B., Ganguly, A. and Majumder, H. K. (2007). Leishmania donovani: dyskinetoplastid cells survive and proliferate in the presence of pyruvate and uridine but do not undergo apoptosis after treatment with camptothecin. Experimental Parasitology 115, 215–219. doi: 10.1016/j.exppara.2006.08.005.CrossRefGoogle Scholar
Sen, N., Das, B. B., Ganguly, A., Banerjee, B., Sen, T. and Majumder, H. K. (2006). Leishmania donovani: intracellular ATP level regulates apoptosis-like death in luteolin induced dyskinetoplastid cells. Experimental Parasitology 114, 204–214. doi: 10.1016/j.exppara.2006.03.013.CrossRefGoogle ScholarPubMed
Shaha, C. (2006). Apoptosis in Leishmania species & its relevance to disease pathogenesis. The Indian Journal of Medical Research 123, 233–244.Google ScholarPubMed
Simons, V., Morrissey, J. P., Latijnhouwers, M., Csukai, M., Cleaver, A., Yarrow, C. and Osbourn, A. (2006). Dual effects of plant steroidal alkaloids on Saccharomyces cerevisiae . Antimicrobial Agents and Chemotherapy 50, 2732–2740. doi: 10.1128/AAC.00289-06.CrossRefGoogle ScholarPubMed
Urbina, J. A., Vivas, J., Lazardi, K., Molina, J., Payares, G., Piras, M. M. and Piras, R. (1996). Antiproliferative effects of delta 24(25) sterol methyl transferase inhibitors on Trypanosoma (Schizotrypanum) cruzi: in vitro and in vivo studies. Chemotherapy 42, 294–307. doi: 10.1159/000239458.CrossRefGoogle Scholar
Vannier-Santos, M. A., Urbina, J. A., Martiny, A., Neves, A. and de Souza, W. (1995). Alterations induced by the antifungal compounds ketoconazole and terbinafine in Leishmania . Journal of Eukaryotic Microbiology 42, 337–346. doi: 10.1111/j.1550-7408.1995.tb01591.x.CrossRefGoogle ScholarPubMed
Vivas, J., Urbina, J. A., and de Souza, W. (1996). Ultrastructural alterations in Trypanosoma (Schizotrypanum) cruzi induced by Δ24(25)-sterol methyltransferase inhibitors and their combinations with ketoconazole. International Journal of Antimicrobial Agents 7, 235–240. doi: org/10.1016/S0924-8579(96)00325-1.CrossRefGoogle Scholar
Vivas, J., Urbina, J. A. and de Souza, W. (1997). Ultrastructural alterations in Trypanosoma (Schizotrypanum) cruzi induced by Delta(24(25)) sterol methyl transferase inhibitors and their combinations with ketoconazole. International Journal of Antimicrobial Agents 8, 1–6. doi: 10.1016/S0924-8579(96)00345-7.CrossRefGoogle Scholar
Warren, L. G. (1960). Metabolism of Schizotrypanum cruzi chagas. I. Effect of culture age and substrate concentration on repiratory rate. The Journal of Parasitology 46, 529–530.CrossRefGoogle Scholar
World Health Organization (2011). Control of the leishmaniases. WHO Technical Report Series No. 949. World Health Organization, Geneva, Switzerland.Google Scholar
Yang, N. C., Ho, W. M., Chen, Y. H. and Hu, M. L. (2002). A convenient one-step extraction of cellular ATP using boiling water for the luciferin-luciferase assay of ATP. Analytical Biochemistry 306, 323–327. doi: 10.1006/abio.2002.5698.CrossRefGoogle ScholarPubMed