Hostname: page-component-586b7cd67f-2plfb Total loading time: 0 Render date: 2024-11-26T20:59:24.239Z Has data issue: false hasContentIssue false
  • English
  • Français

Arrested growth of Trypanosoma cruzi by the calpain inhibitor MDL28170 and detection of calpain homologues in epimastigote forms

Published online by Cambridge University Press:  02 March 2009

L. S. SANGENITO ,
V. ENNES-VIDAL ,
F. A. MARINHO ,
F. F. DA MOTA ,
A. L. S. SANTOS ,
C. M. D'AVILA-LEVY  and
M. H. BRANQUINHA
L. S. SANGENITO
Affiliation:
Departamento de Microbiologia Geral, Instituto de Microbiologia Prof. Paulo de Góes, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
V. ENNES-VIDAL
Affiliation:
Laboratório de Biologia Molecular e Doenças Endêmicas, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, Brazil
F. A. MARINHO
Affiliation:
Departamento de Microbiologia Geral, Instituto de Microbiologia Prof. Paulo de Góes, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
F. F. DA MOTA
Affiliation:
Laboratório de Genômica Funcional e Bioinformática e Laboratório de Biologia Computacional e Sistemas, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, Brazil
A. L. S. SANTOS
Affiliation:
Departamento de Microbiologia Geral, Instituto de Microbiologia Prof. Paulo de Góes, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
C. M. D'AVILA-LEVY
Affiliation:
Laboratório de Biologia Molecular e Doenças Endêmicas, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, Brazil
M. H. BRANQUINHA*
Affiliation:
Departamento de Microbiologia Geral, Instituto de Microbiologia Prof. Paulo de Góes, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
*
*Corresponding author: Departamento de Microbiologia Geral, Instituto de Microbiologia Prof. Paulo de Góes, Universidade Federal do Rio de Janeiro (UFRJ), Centro de Ciências da Saúde (CCS), Av. Carlos Chagas Filho, 373, Bloco I, Cidade Universitária, Rio de Janeiro, RJ, 21941-902, Brazil. Tel: +55 21 2562 6743. Fax: +55 21 2560 8344. E-mail: [email protected]
Get access Rights & Permissions [Opens in a new window]

Summary

In this paper, we aimed to explore the effects of the calpain inhibitor III (MDL28170) and to detect calpain-like molecules (CALPs) in epimastigote forms of Trypanosoma cruzi isolate Dm28c. MDL28170 at 70 μM promoted a powerful reduction in the growth rate after 48 h. The IC50 value was calculated to be 31·7 μM. This inhibitor promoted an increase in the cellular volume, but not cell lysis, resulting in a trypanostatic effect. T. cruzi CALPs presented a strong cross-reactivity with anti-Drosophila melanogaster calpain and anti-cytoskeleton-associated protein from Trypanosoma brucei antibodies, and labelling was found mainly intracellularly. Furthermore, an 80 kDa reactive protein was detected by Western blotting assays. No significant cross-reactivity was found with anti-human brain calpain antibody. The expression of CALPs was decreased in cells kept for long periods in axenic cultures in comparison to a strain recently isolated from mice, as well as in MDL28170-treated cells, the latter being paralleled by an increased expression of cruzipain. Different levels of CALPs expression were also detected in distinct phylogenetic lineages, like Y strain (lineage TCI), Dm28c (TCII) and INPA6147 strain (Z3 zymodeme). These results may contribute for the investigation of the functions of CALPs in trypanosomatids.

Keywords

Trypanosoma cruzicalpainpeptidaseinhibitor
Type
Research Article
Information
Parasitology , Volume 136 , Issue 4 , April 2009 , pp. 433 - 441
Copyright
Copyright © 2009 Cambridge University Press

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

REFERENCES

Andrade, H. M., Murta, S. M. F., Chapeaurouge, A., Perales, J., Nirdé, P. and Romanha, A. (2008). Proteomic analysis of Trypanosoma cruzi resistance to benznidazole. Journal of Proteome Research 7, 2357–2367.CrossRefGoogle ScholarPubMed
Battaglia, F., Trinchese, F., Liu, S., Walter, S., Nixon, R. A. and Arancio, O. (2003). Calpain inhibitors, a treatment for Alzheimer's disease: position paper. Journal of Molecular Neurosciences 20, 357–362.Google ScholarPubMed
Cazzulo, J. J., Stoka, V. and Turk, V. (2001). The major cysteine proteinase of Trypanosoma cruzi: a valid target for chemotherapy of Chagas disease. Current Pharmacological Design 7, 1143–1156.CrossRefGoogle Scholar
Contreras, V. T., Lima, A. R. and Zorrilla, G. (1998). Trypanosoma cruzi: maintenance in culture modify gene and antigenic expression of metacyclic trypomastigotes. Memórias do Instituto Oswaldo Cruz 93, 753–760.CrossRefGoogle ScholarPubMed
d'Avila-Levy, C. M., Araújo, F. M., Vermelho, A. B., Soares, R. M. A., Santos, A. L. S. and Branquinha, M. H. (2005). Proteolytic expression in Blastocrithidia culicis: influence of the endosymbiont and similarities with the virulence factors of pathogenic trypanosomatids. Parasitology 130, 413–420.CrossRefGoogle ScholarPubMed
d'Avila-Levy, C. M., Marinho, F. A., Santos, L. O., Martins, J. L. M., Santos, A. L. S. and Branquinha, M. H. (2006 a). Antileishmanial activity of MDL28170, a potent calpain inhibitor. International Journal of Antimicrobial Agents 28, 138–142.Google ScholarPubMed
d'Avila-Levy, C. M., Dias, F. A., Nogueira de Melo, A. C., Martins, J. L., Lopes, A. H. C. S., Santos, A. L. S., Vermelho, A. B. and Branquinha, M. H. (2006 b). Insights into the role of gp63-like proteins in lower trypanosomatids. FEMS Microbiology Letters 254, 149–156.CrossRefGoogle Scholar
d'Avila-Levy, C. M., Souza, R. F., Gomes, R. C., Vermelho, A. B. and Branquinha, M. H. (2003). A novel extracellular cysteine proteinase from Crithidia deanei. Archives of Biochemistry and Biophysics 420, 1–8.CrossRefGoogle ScholarPubMed
Emori, Y. and Saigo, K. (1994). Calpain localization changes in coordination with actin-related cytoskeletal changes during early embryonic development of Drosophila. The Journal of Biological Chemistry 269, 25137–25142.CrossRefGoogle ScholarPubMed
Engel, J. C., Doyle, P. S., Palmer, J., Hsieh, I., Bainton, D. F. and McKerrow, J. H. (1998). Cysteine protease inhibitors alter Golgi complex ultrastructure and function in Trypanosoma cruzi. The Journal of Cell Science 111, 597–606.CrossRefGoogle ScholarPubMed
Engel, J. C., Torres, C., Hsieh, I., Doyle, P. S. and McKerrow, J. H. (2000). Upregulation of the secretory pathway in cysteine protease inhibitor-resistant Trypanosoma cruzi. The Journal of Cell Science 113, 1345–1354.CrossRefGoogle ScholarPubMed
Ersfeld, K., Barraclough, H. and Gull, K. (2005). Evolutionary relationships and protein domain architecture in an expanded calpain superfamily in kinetoplastid parasites. The Journal of Molecular Evolution 61, 742–757.CrossRefGoogle Scholar
Giese, V., Dallagiovanna, B., Marchini, F. K., Pavoni, D. P., Krieger, M. A. and Goldenberg, S. (2008). Trypanosoma cruzi: a stage-specific calpain-like protein is induced after various kinds of stress. Memórias do Instituto Oswaldo Cruz 103, 598–601.CrossRefGoogle ScholarPubMed
Goll, D. E., Thompson, V. F., Li, H., Wei, W. and Cong, J. (2003). The calpain system. Physiological Reviews 83, 731–801.Google ScholarPubMed
Grynspan, F., Griffin, W. R., Cataldo, A., Katayama, S. and Nixon, R. A. (1997). Active site-directed antibodies identify calpain II as an early-appearing and pervasive component of neurofibrillary pathology in Alzheimer's disease. Brain Research 763, 145–158.CrossRefGoogle ScholarPubMed
Hayes, R. L., Wang, K. K., Kampfl, A., Posmantur, R. M., Newcomb, J. K. and Clifton, G. L. (1998). Potential contribution of proteases to neuronal damage. Drug News Perspectives 11, 215–222.Google ScholarPubMed
Hertz-Fowler, C., Ersfeld, K. and Gull, K. (2001). CAP5.5, a life-cycle-regulated, cytoskeleton-associated protein is a member of a novel family of calpain-related proteins in Trypanosoma brucei. Molecular and Biochemical Parasitology 116, 25–34.CrossRefGoogle ScholarPubMed
Lowry, O. H., Roseborough, N. J., Farr, A. L. and Randall, R. J. (1951). Protein measurement with the Folin phenol reagent. The Journal of Biological Chemistry 193, 264–275.CrossRefGoogle ScholarPubMed
Mendonça-Filho, R. R., Rodrigues, I. A., Alviano, D. S., Santos, A. L. S., Soares, R. M. A., Alviano, C. S., Lopes, A. H. C. S. and Rosa, M. S. (2004). Leishmanicidal activity of polyphenolic-rich extract from husk fiber of Cocos nucifera Linn. (Palmae). Research in Microbiology 155, 136–143.CrossRefGoogle ScholarPubMed
Miles, M. A., Souza, A., Povoa, M., Shaw, J. J., Lainson, R. and Toye, P. J. (1978). Isozymic heterogeneity of Trypanosoma cruzi in the first autochtonous patients with Chagas’ disease in Amazonian Brazil. Nature, London 272, 819–821.CrossRefGoogle Scholar
Pereira, F. M., Elias, C. G. R., d'Avila-Levy, C. M., Branquinha, M. H. and Santos, A. L. S. (2009). Cysteine peptidases in Herpetomonas samuelpessoai are modulated by temperature and dimethylsulfoxide-triggered differentiation. Parasitology 136, 45–54.CrossRefGoogle ScholarPubMed
Rami, A., Ferger, D. and Krieglstein, J. (1997). Blockade of calpain proteolytic activity rescues neurons from glutamate excitotoxicity. Neuroscience Research 27, 93–97.CrossRefGoogle ScholarPubMed
Ramos, C. S., Franco, F. A. L., Smith, D. F. and Uliana, S. R. B. (2004). Characterisation of a new Leishmania META gene and genomic analysis of the META cluster. FEMS Microbiology Letters 238, 213–219.Google ScholarPubMed
Salotra, P., Duncan, R. C., Singh, R., Raju, B. V. S., Sreenivas, G. and Nakhasi, H. L. (2006). Upregulation of surface proteins in Leishmania donovani isolated from patients of post kala-azar dermal leishmaniasis. Microbes and Infection 8, 637–644.CrossRefGoogle ScholarPubMed
Santos, A. L. S., d'Avila-Levy, C. M., Dias, F. A., Ribeiro, R. O., Pereira, F. M., Elias, C. G., Souto-Padrón, T., Lopes, A. H., Alviano, C. S., Branquinha, M. H. and Soares, R. M. A. (2006). Phytomonas serpens: cysteine peptidase inhibitors interfere with growth, ultrastructure and host adhesion. International Journal for Parasitology 36, 47–56.CrossRefGoogle ScholarPubMed
Saxena, A., Worthey, E. A., Yan, S., Leland, A., Stuart, K. D. and Myler, P. J. (2003). Evaluation of differential gene expression in Leishmania major Friedlin procyclics and metacyclics using DNA microarray analysis. Molecular and Biochemical Parasitology 129, 103–114.CrossRefGoogle ScholarPubMed
Souto, R. P., Fernandes, O., Macedo, A. M., Campbell, D. A. and Zingales, B. (1996). DNA markers define two major phylogenetic lineages of Trypanosoma cruzi. Molecular and Biochemical Parasitology 83, 141–152.CrossRefGoogle ScholarPubMed
Tomas, A. M., Miles, M. A. and Kelly, J. M. (1997). Overexpression of cruzipain, the major cysteine proteinase of Trypanosoma cruzi, is associated with enhanced metacyclogenesis. European Journal of Biochemistry 244, 596–603.CrossRefGoogle ScholarPubMed
Troeberg, L., Morty, R. E., Pike, R. N., Lonsdale-Eccles, J. D., Palmer, J. T., McKerrow, J. H. and Coetzer, T. H. (1999). Cysteine proteinase inhibitors kill cultured bloodstream forms of Trypanosoma brucei brucei. Experimental Parasitology 91, 349–355.CrossRefGoogle ScholarPubMed
Vergnes, B., Gourbal, B., Gorard, I., Sundar, S., Drummelsmith, J. and Ouellette, M. (2007). A proteomics screen implicates HSP83 and a small kinetoplastid calpain-related protein in drug resistance in Leishmania donovani clinical field isolates by modulating drug-induced programmed cell death. Molecular Cell Proteomics 6, 88–101.CrossRefGoogle Scholar
Vermelho, A. B., Giovanni-de-Simone, S., d'Avila-Levy, C. M., Santos, A. L. S., Melo, A. C. N., Silva, F. P. Jr,, Bom, E. P. S. and Branquinha, M. H. (2007). Trypanosomatidae peptidases: a target for drugs development. Current Enzyme Inhibition 3, 19–48.Google Scholar
World Health Organization (2005). Infectious Diseases Home Burdens and Trends. Available from: <http://www.who.int/ctd/chagas/burdens.htm>..>Google Scholar
Yong, V., Schmitz, V., Vannier-Santos, M. A., Lima, A. P. C. A., Lalmanach, G., Juliano, L., Gauthier, F. and Scharfstein, J. (2000). Altered expression of cruzipain and a cathepsin B-like target in a Trypanosoma cruzi cell line displaying resistance to synthetic inhibitors of cysteine-proteinases. Molecular and Biochemical Parasitology 109, 47–59.Google Scholar