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Effects of serine protease inhibitors on viability and morphology of Leishmania (Leishmania) amazonensis promastigotes

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Abstract

To investigate the importance of serine proteases in Leishmania amazonensis promastigotes, we analyzed the effects of classical serine protease inhibitors and a Kunitz-type inhibitor, obtained from sea anemone Stichodactyla helianthus (ShPI-I), on the viability and morphology of parasites in culture. Classical inhibitors were selected on the basis of their ability to inhibit L. amazonensis serine proteases, previously described. The N-tosyl-l-phenylalanine chloromethyl ketone (TPCK) and benzamidine (Bza) inhibitors, which are potential Leishmania proteases inhibitors, in all experimental conditions reduced the parasite viability, with regard to time dependence. On the other hand, N-tosyl-lysine chloromethyl ketone (TLCK) did not significantly affect the parasite viability, as it was poor Leishmania enzymes inhibitor. Ultrastructural analysis demonstrated that both Bza and TPCK induced changes in the flagellar pocket region with membrane alteration, including bleb formation. However, TPCK effects were more pronounced than those of Bza in Leishmania flagellar pocket in plasma membrane, and intracellular vesicular bodies was visualized. ShPI-I proved to be a powerful inhibitor of L. amazonensis serine proteases and the parasite viability. The ultrastructural alterations caused by ShPI-I were more dramatic than those induced by the classical inhibitors. Vesiculation of the flagellar pocket membrane, the appearance of a cytoplasmic vesicle that resembles an autophagic vacuole, and alterations of promastigotes shape resulted.

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Abbreviations

BHI:

brain heart infusion

BPTI:

bovine pancreatic trypsin inhibitor

Bza:

benzamidine

IC:

inhibitor concentration

EM:

electron microscopy

MTT:

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide

ShPI-I:

Kunitz-type protease inhibitor from sea anemone Stichodactyla helianthus

L- TAME:

N-ρ-tosyl-l-arginine methyl ester

TLCK:

N-tosyl-lysine chloromethyl ketone

TPCK:

N-tosyl-l-phenylalanine chloromethyl ketone

References

  • Antuch W, Berndt KD, Chavéz MA, Delfín J, Wüthrich K (1993) The NMR solution structure of a Kunitz-type proteinase inhibitor from the sea anemone Stichodactyla helianthus. Eur J Biochem 212:675–684

    Article  PubMed  CAS  Google Scholar 

  • Barankiewicz J, Dosh HM, Cohen A (1988) Extracellular nucleotide catabolism in human B and T lymphocytes. J Biol Chem 263:7094–7098

    PubMed  CAS  Google Scholar 

  • Bera A, Singh S, Nagaraj R, Vaidya T (2003) Induction of autophagic cell death in Leishmania donovani by antimicrobial peptides. Mol Biochem Parasitol 127:23–35

    Article  PubMed  CAS  Google Scholar 

  • Biederbick A, Kern HF, Elsasser HP (1995) Monodansylcadaverine (MDC) is a specific in vivo marker for autophagic vacuoles. Eur J Cell Biol 66:3–14

    PubMed  CAS  Google Scholar 

  • Conseil V, Soete M, Dubremetz JF (1999) Serine protease inhibitors block invasion of host cells by Toxoplasma gondii. Antimicrob Agents Chemother 46:1358–1361

    Google Scholar 

  • Coombs GH, Mottram JC (1997). Proteases in trypanosomatids. In: Hide G, Mottram JC, Coombs GH, Holmes PH (eds) Trypanosomiasis and leishmaniasis. CAB International, London, pp 176–197

    Google Scholar 

  • Croft SL, Coombs GH (2003) Leishmaniasis-current chemotherapy and recent advances in the search for novel drugs. Trends Parasitol 19:502–508

    Article  PubMed  CAS  Google Scholar 

  • Delfin J, Martinez I, Antuch W, Morera V, Gonzalez Y, Rodriguez R, Marquez M, Saroyan A, Larionova N, Diaz J, Padron G, Chavez M (1996) Purification, characterization and immobilization of proteinase inhibitors from Stichodactyla helianthus. Toxicon 34:1367–1376

    Article  PubMed  CAS  Google Scholar 

  • Desai PV, Patny A, Sabnis Y, Tekwani B, Gut J, Rosenthal P, Srivastava A, Avery M (2004) Identification of novel parasitic cysteine protease inhibitors using virtual screening. 1. The ChemBridge database. J Med Chem 16:6609–6615

    Article  CAS  Google Scholar 

  • Herwaldt BL (1999) Leishmaniasis. Lancet 354:1191–1199

    Article  PubMed  CAS  Google Scholar 

  • Kano SI, Shouji A, Asou K, Ishikawa M (2003) Effects of naringin on hydrogen peroxide-induced cytotoxicity and apoptosis in P388 Cells. J Pharmacol Sci 92:166–170

    Article  Google Scholar 

  • Krowarsch D, Cierpicki T, Jelen F, Otlewski J (2003) Canonical protein inhibitors of serine proteases. Cell Mol Life Sci 60:2427–2444

    Article  PubMed  CAS  Google Scholar 

  • Lazardi K, Urbina JA, De Souza W (1991) Ultrastructural alterations induced by two ergosterol biosynthesis inhibitors, ketoconazole and terbinafine, on epimastigotes and amastigotes of Trypanosoma (schizotrypanum) cruzi. Antimicrob Agents Chemother 34:2097–2105

    Google Scholar 

  • McKerrow JH, Engel JC, Caffrey CR (1999) Cysteine protease inhibitors as chemotherapy for parasitic infections. Bioorg Med Chem 7:639–644

    Article  PubMed  CAS  Google Scholar 

  • Melendez A, Talloczy Z, Seaman M, Eskelinen EL, Hall DH, Levine B (2003) Autophagy genes are essential for dauer development and life-span extension in C. elegans. Science 301:1387–1391

    Article  PubMed  CAS  Google Scholar 

  • Mizushima N (2004) Methods for monitoring autophagy. Int J Biochem Cell Biol 36:2491–2502

    Article  PubMed  CAS  Google Scholar 

  • Mizushima N, Yamamoto A, Matsui M, Yoshimori T, Ohsumi Y (2004) In vivo analysis of autophagy in response to nutrient starvation using transgenic mice expressing a fluorescent autophagosome marker. Mol Biol Cell 15:1101–1111

    Article  PubMed  CAS  Google Scholar 

  • Morgado-Díaz JA, Silva-Lopez RE, Alves CR, Soares MJ, Corte-Real S, Giovanni-De-Simone S (2005) Subcellular localization of an intracellular serine protease of 68 kDa in Leishmania (Leishmania) amazonensis promastigotes. Mem Inst Oswaldo Cruz 100:377–383

    Article  PubMed  Google Scholar 

  • O’Daly JA, Cabrera CZ (1986) Immunization of hamsters with TLCK-killed parasites induces protection against Leishmania infection. Acta Trop 43:225–236

    PubMed  CAS  Google Scholar 

  • Otlewski J, Krowarsch D, Apostoluk W (1999) Protein inhibitors of serine proteinases. Acta Biochim Pol 46:531–565

    PubMed  CAS  Google Scholar 

  • Paris C, Loiseau PM, Bories C, Bréard J (2004) Miltefosine Induces Apoptosis-Like Death in Leishmania donovani Promastigotes. Antimicrob Agents Chemother 48:852–859

    Article  PubMed  CAS  Google Scholar 

  • Powers JC, Asgian JL, Ekici OD, James KE (2002) Irreversible inhibitors of serine, cysteine, and threonine proteases. Chem Rev 102:4639–4750

    Article  PubMed  CAS  Google Scholar 

  • Ribeiro de Andrade A, Santoro MM, Norma de Melo M, Mares-Guia M (1998). Leishmania (Leishmania) amazonensis: purification and enzyme characterization of a soluble serine oligopeptidase from promastigotes. Exp Parasitol 89:153–160

    Article  Google Scholar 

  • Roggwille E, Bétoulle MEM, Blisnick T, Braun-Breton C (1996) A role for erythrocyte band 3 degradation by the parasite gp 76 serine protease in the formation of the parasitophorus vacuole during invasion of erythrocytes by Plasmodium falciparum. Mol Biochem Parasitol 82:13–24

    Article  Google Scholar 

  • Rosenthal PJ (1999) Proteases of protozoan parasites. Adv Parasitol 43:105–159

    Article  PubMed  CAS  Google Scholar 

  • Sadij M, McKerrow JH (2002) Cysteine proteases of parasitic organisms. Mol Biochem Parasitol 120:1–21

    Article  Google Scholar 

  • Savoia D, Avanzini C, Allice T, Callone E, Guella G, Dini F (2004) Antimicrobial activity of euplotin C, the sesquiterpene taxonomic marker from the marine ciliate Euplotes crassus. Antimicrob Agents Chemother 48:3828–3833

    Article  PubMed  CAS  Google Scholar 

  • Selzer PM, Pingel S, Hsieh Y, Ugeles B, Chan VJ, Engel JC, Bogyo M, Russelli DG, Sakanari JA, Sakanari J, Mckerrow JH (1999) Cysteine protease inhibitors as chemotherapy: lessons from a parasite target. Proc Natl Acad Sci U S A 96:11015–11022

    Article  PubMed  CAS  Google Scholar 

  • Shaw MK, Roos DS, Tilney LG (2002) Cysteine and serine protease inhibitors block intracellular development and disrupt the secretory pathway of Toxoplasma gondii. Microbes Infect 4:119–132

    Article  PubMed  CAS  Google Scholar 

  • Silva-Lopez RE, Giovanni De Simone S (2004a) A serine protease from a detergent-soluble extract of Leishmania (Leishmania) amazonensis. Z Naturforsch 59c:590–598

    Google Scholar 

  • Silva-Lopez RE, Giovanni De Simone S (2004b) Leishmania (Leishmania) amazonensis: purification and characterization of a promastigote serine protease. Exp Parasitol 107:173–182

    Article  PubMed  CAS  Google Scholar 

  • Silva-Lopez RE, Morgado-Díaz JA, Alves CR, Côrte-Real S, Giovanni De Simone S (2004) Subcellular localization of an extracellular serine protease in Leishmania (Leishmania) amazonensis. Parasitol Res 93:328–331

    Article  PubMed  CAS  Google Scholar 

  • Silva-Lopez RE, Pinto-Coelho MG, Giovanni De Simone S (2005) Characterization of an extracellular serine protease of Leishmania (Leishmania) amazonensis. Parasitol 131:85–96

    Article  CAS  Google Scholar 

  • Sturzebecher J, Vieweg H, Wikstrom P, Turk D, Bode W (1992) Interactions of thrombin with benzamidine-based inhibitors. Biol Chem Hoppe-Seyler 373:491–506

    PubMed  CAS  Google Scholar 

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Acknowledgments

This work was supported with grants from Fundação de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ), Fundação Oswaldo Cruz (FIOCRUZ), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), and Fundação Ary Frauzino para Pesquisa e Controle do Câncer (FAF). We are grateful to Mr. Genival Alves de Souza and Marcelo Tanaka for technical assistance on Leishmania cultures and electron microscopy and Angel Ramirez for enzyme kinetic advices. This manuscript was revised by Mitchell Raymond Lishan, native of Chicago, IL, USA.

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Authors and Affiliations

  1. Laboratório de Bioquímica de Proteínas e Peptídeos, Departamento de Bioquímica e Biologia Molecular, Instituto Oswaldo Cruz, FIOCRUZ, Av. Brasil 4365, 21045-900, Rio de Janeiro, RJ, Brazil

    R. E. Silva-Lopez & S. Giovanni-De-Simone

  2. Divisão de Biologia Celular, Pesquisa Básica, Instituto Nacional de Câncer, Rio de Janeiro, RJ, Brazil

    J. A. Morgado-Díaz

  3. Centro de Estudio de Proteínas, Facultad de Biologia, Universidad de la Habana, Havana, Cuba

    M. A. Chávez

  4. Departamento de Biologia Celular e Molecular, Instituto de Biologia, Universidade Federal Fluminense, Niterói, RJ, Brazil

    S. Giovanni-De-Simone

Authors
  1. R. E. Silva-Lopez
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  2. J. A. Morgado-Díaz
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  3. M. A. Chávez
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  4. S. Giovanni-De-Simone
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Correspondence to R. E. Silva-Lopez.

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Silva-Lopez, R.E., Morgado-Díaz, J.A., Chávez, M.A. et al. Effects of serine protease inhibitors on viability and morphology of Leishmania (Leishmania) amazonensis promastigotes. Parasitol Res 101, 1627–1635 (2007). https://doi.org/10.1007/s00436-007-0706-5

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  • DOI: https://doi.org/10.1007/s00436-007-0706-5

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