Hostname: page-component-848d4c4894-pjpqr Total loading time: 0 Render date: 2024-06-19T14:42:53.342Z Has data issue: false hasContentIssue false
  • English
  • Français

Dissimilar peptidase production by avirulent and virulent promastigotes of Leishmania braziliensis: inference on the parasite proliferation and interaction with macrophages

Published online by Cambridge University Press:  27 July 2009

A. K. C. LIMA ,
C. G. R. ELIAS ,
J. E. O. SOUZA ,
A. L. S. SANTOS  and
P. M. L. DUTRA
A. K. C. LIMA
Affiliation:
Disciplina de Parasitologia, Departamento de Microbiologia, Imunologia e Parasitologia (DMIP), Faculdade de Ciências Médicas (FCM), Universidade do Estado do Rio de Janeiro (UERJ), Rio de Janeiro, Brazil
C. G. R. ELIAS
Affiliation:
Laboratório de Estudos Integrados em Bioquímica Microbiana, Departamento de Microbiologia Geral, Instituto de Microbiologia Prof. Paulo de Góes (IMPPG), Centro de Ciências da Saúde (CCS), Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, Brazil
J. E. O. SOUZA
Affiliation:
Disciplina de Parasitologia, Departamento de Microbiologia, Imunologia e Parasitologia (DMIP), Faculdade de Ciências Médicas (FCM), Universidade do Estado do Rio de Janeiro (UERJ), Rio de Janeiro, Brazil
A. L. S. SANTOS
Affiliation:
Laboratório de Estudos Integrados em Bioquímica Microbiana, Departamento de Microbiologia Geral, Instituto de Microbiologia Prof. Paulo de Góes (IMPPG), Centro de Ciências da Saúde (CCS), Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, Brazil
P. M. L. DUTRA*
Affiliation:
Disciplina de Parasitologia, Departamento de Microbiologia, Imunologia e Parasitologia (DMIP), Faculdade de Ciências Médicas (FCM), Universidade do Estado do Rio de Janeiro (UERJ), Rio de Janeiro, Brazil
*
*Corresponding author: Departamento de Microbiologia, Imunologia e Parasitologia, Rua Prof. Manoel de Abreu, 444, PAPC 5° andar, Vila Isabel, Rio de Janeiro, RJ 20550-170, Brazil. Tel: +55 21 2587 6487. Fax: +55 21 2587 6148. E-mail: pdutra@uerj.br
Get access Rights & Permissions [Opens in a new window]

Summary

In the present paper, we have analysed the cellular and extracellular proteolytic activity profiles in 2 distinct Leishmania braziliensis strains: a recently isolated (virulent) and a laboratory-adapted (avirulent) strain. Quantitative and qualitative differences on the peptidase expression were observed in both strains. For instance, low-molecular mass acidic cysteine peptidase activities were detected exclusively in the virulent strain. Similarly, metallopeptidase activities were mainly produced by L. braziliensis virulent promastigotes. Interestingly, metallo- and cysteine peptidase activities were drastically reduced after several in vitro passages of the virulent strain. Western blotting, flow cytometry and fluorescence microscopy analyses were performed to detect homologous of the major leishmania metallopeptidase (gp63) and cysteine peptidase (cpb) in virulent and avirulent strains of L. braziliensis. Our results revealed that the virulent strain produced higher amounts of gp63 and cpb molecules, detected both in the surface and cytoplasm regions, than the avirulent counterpart. Metallo- (1,10-phenanthroline and EGTA) and cysteine peptidase (E-64) inhibitors arrested the growth of L. braziliensis virulent strain in a dose-dependent manner, as well as the association index with peritoneal murine macrophages. Conversely, these peptidase inhibitors did not affect either the proliferation or the cellular interaction of the avirulent strain. Corroborating these findings, the pre-treatment of the virulent strain with both anti-peptidase antibodies promoted a prominent reduction in the interaction with macrophages, while the association index of the avirulent strain to macrophage was only slightly diminished. Moreover, the spent culture medium from virulent strain significantly enhanced the association index between avirulent strain and macrophages, and this effect was reversed by 1,10-phenanthroline. Collectively, the results presented herein suggest that peptidases participate in several crucial processes of L. braziliensis.

Keywords

Leishmania braziliensispeptidasesproteolytic inhibitorsgrowthcellular interactiongp63cpb
Type
Research Article
Information
Parasitology , Volume 136 , Issue 10 , September 2009 , pp. 1179 - 1191
Copyright
Copyright © Cambridge University Press 2009

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

Alexander, J., Coombs, G. H. and Mottram, J. C. (1998). Leishmania mexicana cysteine proteinase-deficient mutants have attenuated virulence for mice and potentiate a Th1 response. The Journal of Immunology 161, 6794–6801.Google Scholar
Bart, G., Frame, M. J., Carter, R., Coombs, G. H. and Mottram, J. C. (1997). Cathepsin B-like cysteine proteinase-deficient mutants of Leishmania mexicana. Molecular and Biochemical Parasitology 88, 53.Google Scholar
Bates, P. A., Robertson, C. D. and Coombs, G. H. (1994). Expression of cysteine proteinases by metacyclic promastigotes of Leishmania mexicana. Journal of Eukaryotic Microbiology 41, 199–203.CrossRefGoogle ScholarPubMed
Bonaldo, M. C., D'Escoffier, L. N., Salles, J. M. and Goldenberg, S. (1991). Characterization and expression of protease during Trypanosoma cruzi metacyclogenesis. Experimental Parasitology 73, 44–51.Google Scholar
Bouvier, J., Schneider, P., Etges, R. and Bordier, C. (1990). Peptide substrate specificity of the membrane bound metalloprotease of Leishmania. Biochemistry 29, 10113–10119.Google Scholar
Brittingham, A., Miller, M. A., Donelson, J. E. and Wilson, M. E. (2001). Regulation of GP63 mRNA stability in promastigotes of virulent and attenuated Leishmania chagasi. Molecular and Biochemical Parasitology 112, 51–59.CrossRefGoogle ScholarPubMed
Chakraborty, R., Chakraborty, P. and Basu, M. K. (1998). Macrophage mannosyl fucosyl receptor: its role in invasion of virulent and avirulent Leishmania donovani promastigotes. Bioscience Reports 18, 129–142.Google Scholar
Chaudhuri, G. and Chang, K. P. (1988). Acid protease activity of major promastigotes. Molecular and Biochemical Parasitology 27, 43–52.CrossRefGoogle Scholar
Cuervo, P., Sabóia-Vahia, L., Silva-Filho, F. C., Fernandes, O., Cupolillo, E. and Jesus, J. B. (2006). A zymographic study of metalloprotease activities in extracts and extracellular secretions of Leishmania (Viannia) braziliensis strains. Parasitology 132, 177–185.CrossRefGoogle ScholarPubMed
Cuervo, P., Santos, A. L. S., Alves, C. R., Menezes, G. C., Silva, B. A., Britto, C., Fernandes, O., Cupolillo, E. and Jesus, J. B. (2008). Cellular localization and expression of gp63 homologous metalloproteases in Leishmania (Viannia) braziliensis strains. Acta Tropica 106, 143–148.CrossRefGoogle ScholarPubMed
Denise, H., McNeil, K., Brooks, D. R., Alexander, J., Coombs, G. H. and Mottram, J. C. (2003). Expression of multiple CPB genes encoding cysteine proteases is required for Leishmania mexicana virulence in vivo. Infection and Immunity 71, 3190–3195.CrossRefGoogle ScholarPubMed
Elias, C. G. R., Pereira, F. M., Silva, B. A., Alviano, C. S., Soares, R. M. A. and Santos, A. L. S. (2006). Leishmanolysin (gp63 metallopeptidase)-like activity extracellularly released by Herpetomonas samuel pessoai. Parasitology 132, 37–47.CrossRefGoogle Scholar
Frame, M. J., Mottram, J. C. and Coombs, G. H. (2000). Analysis of the roles of cysteine proteinases of Leishmania mexicana in the host–parasite interaction. Parasitology 121, 367–377.CrossRefGoogle ScholarPubMed
Garcia, M. R., Graham, S., Harris, R. A., Beverlwy, S. M. and Kayne, P. M. (1997). Epitope cleavage by Leishmania endopeptidases(s) limits the efficiency of the exogenous pathway of major histocompatibility complex class I-associated antigen presentation. European Journal of Immunology 27, 1005–1013.CrossRefGoogle ScholarPubMed
Heussen, C. and Dowdle, E. B. (1980). Electrophoretic analysis of plasminogen activators in polyacrilamide gels containing sodium dodecyl sulfate and copolymerized substrates. Analytical Biochemistry 102, 196–202.CrossRefGoogle Scholar
Hey, A. S., Theander, T. G., Hviid, L., Hazrati, S. M., Kemp, M. and Kharazmi, A. (1994). The major surface glycoprotein (gp63) from Leishmania major and Leishmania donovani cleaves CD4 molecules on human T cells. The Journal of Immunology 152, 4542–4548.Google Scholar
Irvine, J. W., North, M. J. and Coombs, G. H. (1997). Use of inhibitors to identify essential cysteine proteinases of Trichomonas vaginalis. FEMS Microbiology Letters 149, 45–50.CrossRefGoogle ScholarPubMed
Joshi, P. B., Kelly, B. L., Kamhawi, S., Sacks, D. L. and McMaster, W. R. (2002). Targeted gene deletion in Leishmania major identifies leishmanolysin (GP63) as a virulence factor. Molecular and Biochemical Parasitology 120, 33–40.Google Scholar
Lanfranco, M. F., Loayza-Muro, R., Clark, D., Núñez, R., Zavaleta, A. I., Jimenez, M., Meldal, M., Coombs, G. H., Mottram, J. C., Izidoro, M., Juliano, M. A., Juliano, L. and Arévalo, J. (2008). Expression and substrate specificity of a recombinant cysteine proteinase B of Leishmania braziliensis. Molecular and Biochemical Parasitology 161, 91–100.CrossRefGoogle ScholarPubMed
Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951). Protein measurement with the folin phenol reagent. The Journal of Biological Chemistry 193, 265–275.Google Scholar
McGwire, B. S., Chang, K. P. and Engman, D. M. (2003). Migration through the extracellular matrix by the parasitic protozoan Leishmania is enhanced by surface metalloprotease gp63. Infection and Immunity 71, 1008–1010.Google Scholar
McKerrow, J. H., Sun, E., Rosenthal, P. J. and Bouvier, J. (1993). The proteases and pathogenicity of parasitic protozoa. Annual Review of Microbiology 47, 821–853.CrossRefGoogle ScholarPubMed
Mottram, J. C., Souza, A. E., Hutchison, J. E., Carter, R., Frame, M. J. and Coombs, G. H. (1996). Evidence from disruption of the Imcpb gene array of Leishmania mexicana that cysteine proteinases are virulence factors. Proceedings of the National Academy of Sciences, USA 93, 6008–6013.Google Scholar
Mottram, J. C., Frame, M. J., Brooks, D. R., Tetley, L., Hutchison, J. E., Souza, A. E. and Coombs, G. H. (1997). The multiple cpb cysteine proteinase genes of Leishmania mexicana encode isoenzymes which differ in their stage-regulation and substrate preferences. The Journal of Biological Chemistry 272, 14285–14293.CrossRefGoogle ScholarPubMed
Mottram, J. C., Brooks, D. R. and Coombs, G. H. (1998). Roles of cysteine proteinases of trypanosomes and Leishmania in host–parasite interactions. Current Opinion in Microbiology 1, 455–460.Google Scholar
Parsons, M. and Ruben, L. (2000). Pathways involved in environmental sensing in trypanosomatids. Parasitology Today 16, 56–62.Google 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.Google Scholar
Sacks, D. and Sher, A. (2002). Evasion of innate immunity by parasitic protozoa. Nature Immunology 3, 1041–1047.CrossRefGoogle ScholarPubMed
Sádlová, J., Volf, P., Victoir, K., Dujardin, J. and Votýpka, J. (2006). Virulent and attenuated lines of Leishmania major: DNA karyotypes and differences in metalloproteinase GP63. Folia Parasitologica 53, 81–90.Google Scholar
Santos, A. L. S., Alviano, C. S. and Soares, R. M. A. (2005). Use of proteolytic enzymes as an additional tool for trypanosomatid identification. Parasitology 130, 79–88.CrossRefGoogle ScholarPubMed
Santos, A. L. S., d'Avila-Levy, C. M., Dias, F. A., Ribeiro, R. O., Pereira, F. M., Elias, C. G. R., Souto-Padrón, T., Lopes, A. H. C. S., 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 Parasitolology 36, 47–56.CrossRefGoogle ScholarPubMed
Segovia, M., Artero, J. M., Mellado, E. and Chance, M. L. (1992). Effects of long-term in vitro cultivation on the virulence of cloned lines of Leishmania major promastigotes. Annals of Tropical Medicine and Parasitology 86, 347–354.CrossRefGoogle ScholarPubMed
Selzer, P. M., Pingel, S., Hsieh, I., Ugele, B., Chan, V. J., Engel, J. C., Bogyo, M., Russel, D. G., Sakanari, J. A. and McKerrow, J. H. (1999). Cysteine protease inhibitors as chemotherapy: lessons from parasite target. Proceedings of the National Academy of Sciences, USA 96, 11015–11022.CrossRefGoogle ScholarPubMed
Soares, R. M. A., Santos, A. L. S., Bonaldo, M. C., Andrade, A. F. B., Alviano, C. S., Angluster, J. and Goldenberg, S. (2003). Leishmania (Leishmania) amazonensis: differential expression of proteinases and cell-surface polypeptides in avirulent and virulent promastigotes. Experimental Parasitology 104, 104–112.Google Scholar
Souza, A. E., Waugh, S., Coombs, G. H. and Mottram, J. C. (1992). Characterization of a multicopy gene for a major stage-specific cysteine proteinase of Leishmania mexicana. FEBS Letters 311, 124–127.CrossRefGoogle Scholar
Troeberg, L., Morty, R. E., Pike, R. N., Lonsdale-Eccles, J. D., Palmer, J. T., McKerrow, J. H. and Coetzer, T. H. T. (1999). Cysteine proteinase inhibitors kill cultured bloodstream forms of Trypanosoma brucei brucei. Experimental Parasitology 91, 349–355.CrossRefGoogle ScholarPubMed
Vermelho, A. B., Giovanni-de-Simone, S., d'Avila-Levy, C. M., Santos, A. L. S., Nogueira de Melo, A. C., Silva-Junior, F. P., Bom, E. P. and Branquinha, M. H. (2007). Trypanosomatidae peptidases: a target for drugs development. Current Enzyme Inhibition 3, 19–48.Google Scholar
WHO/OMS – World Health Organization/Organisation Mondiale de La Santé (2000). Disease and its Impact. Geographical Distribution. Available from www.who.int/emc/diseases/leish/index.htmlGoogle Scholar
Wilson, M. E., Hardin, K. K. and Donelson, J. E. (1989). Expression of the major surface glycoprotein of Leishmania donovani chagasi in virulent and attenuated promastigotes. The Journal of Immunology 143, 678–684.CrossRefGoogle ScholarPubMed
Yao, C., Donelson, J. E. and Wilson, M. E. (2003). The major surface protease (MSP or GP63) of Leishmania sp. biosynthesis, regulation of expression, and function. Molecular and Biochemical Parasitology 132, 1–16.Google Scholar