Hostname: page-component-78c5997874-lj6df Total loading time: 0 Render date: 2024-11-10T11:58:46.107Z Has data issue: false hasContentIssue false
  • English
  • Français

Leishmanolysin (gp63 metallopeptidase)-like activity extracellularly released by Herpetomonas samuelpessoai

Published online by Cambridge University Press:  21 September 2005

C. G. R. ELIAS ,
F. M. PEREIRA ,
B. A. SILVA ,
C. S. ALVIANO ,
R. M. A. SOARES  and
A. L. S. SANTOS
C. G. R. ELIAS
Affiliation:
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), Ilha do Fundão, Rio de Janeiro, RJ 21941-590, Brazil
F. M. PEREIRA
Affiliation:
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), Ilha do Fundão, Rio de Janeiro, RJ 21941-590, Brazil
B. A. SILVA
Affiliation:
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), Ilha do Fundão, Rio de Janeiro, RJ 21941-590, Brazil
C. S. ALVIANO
Affiliation:
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), Ilha do Fundão, Rio de Janeiro, RJ 21941-590, Brazil
R. M. A. SOARES
Affiliation:
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), Ilha do Fundão, Rio de Janeiro, RJ 21941-590, Brazil
A. L. S. SANTOS
Affiliation:
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), Ilha do Fundão, Rio de Janeiro, RJ 21941-590, Brazil
Get access Rights & Permissions [Opens in a new window]

Abstract

In previous studies, we showed that Herpetomonas samuelpessoai produced a large amount of a surface-located metallopeptidase that presented similar biochemical properties to that of gp63 from Leishmania spp., which is a well-known virulence factor expressed by these digenetic parasites. The present study aims to identify the proteolytic activity released by living H. samuelpessoai cells. In this context, the parasites were incubated in phosphate buffer up to 4 h, and the supernatants were obtained by centrifugation and filtration steps and were then applied on SDS–PAGE to determine the secretory protein profile and on gelatin-SDS–PAGE to identify the proteolytic activity. The results demonstrated that H. samuelpessoai secreted at least 12 polypeptides and an extracellular peptidase of 66 kDa. This enzyme had its activity diminished by 1,10-phenanthroline, EDTA and EGTA. This metallopeptidase was active in a broad spectrum of pH, showing maximum activity at pH 6·0 at 37 °C. Casein was also cleaved by this secretory proteolytic enzyme, while bovine serum albumin and haemoglobin were not degraded under these conditions. Fluorescence microscopy and flow cytometry using anti-gp63 antibody against leishmanolysin of L. amazonensis demonstrated the presence of similar molecules on the cell-surface of H. samuelpessoai. Moreover, immunoblot analysis showed the presence of a reactive polypeptide in the cellular extract and in the supernatant fluid of H. samuelpessoai, which suggests immunological similarities between these two distinct trypanosomatids. The zinc-metallopeptidase inhibitor 1,10-phenanthroline was able to inhibit the secretion of the 66 kDa metallopeptidase in a dose-dependent manner, while the phospholipase C inhibitor (p-CMPS) did not alter the secretion pattern. Additionally, anti-cross-reacting determinant (CRD) antibody failed to recognize any secreted polypeptide from H. samuelpessoai. Collectively, these results suggest that the gp63-like molecule was released from the H. samuelpessoai surface by proteolysis instead of phospholipolysis, in a similar mechanism to that observed in Leishmania.

Keywords

Herpetomonas samuelpessoaileishmanolysinmetallopeptidasessecretiontrypanosomatids
Type
Research Article
Information
Parasitology , Volume 132 , Issue 1 , January 2006 , pp. 37 - 47
Copyright
© 2005 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

Almeida, F. V. S., Branquinha, M. H., Giovanni-de-simone, S. and Vermelho, A. B. ( 2003). Extracellular metalloproteinase activity in Phytomonas françai. Parasitology Research 89, 320–322.Google Scholar
Bangs, J. D., Ransom, D. M., Nimick, M., Christie, G. and Hooper, N. M. ( 2001). In vitro cytocidal effects on Trypanosoma brucei and inhibition of Leishmania major GP63 by peptidomimetric metalloprotease inhibitors. Molecular and Biochemical Parasitology 114, 111–117.CrossRefGoogle 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.CrossRefGoogle Scholar
Brittingham, A., Morrison, C. J., McMaster, W. R., McGwire, B. S., Chang, K. P. and Mosser, D. M. ( 1995). Role of the Leishmania surface protease gp63 in complement fixation, cell adhesion, and resistance to complement-mediated lysis. The Journal of Immunology 155, 3102–3111.CrossRefGoogle Scholar
Chaudhuri, G. and Chang, K. P. ( 1988). Acid protease activity of a major surface membrane glycoprotein (gp63) from Leishmania mexicana promastigotes. Molecular and Biochemical Parasitology 27, 43–52.CrossRefGoogle Scholar
Cuevas, I. C., Cazzulo, J. J. and Sanchez, D. O. ( 2003). gp63 homologues in Trypanosoma cruzi: surface antigens with metalloproteinase activity and a possible role in the host cell infection. Infection and Immunity 71, 5739–5749.CrossRefGoogle Scholar
D'Ávila-levy, C. M., Melo, A. C. N., Vermelho, A. B. and Branquinha, M. H. ( 2001). Differential expression of proteolytic enzymes in endosymbiont-harboring Crithidia species. FEMS Microbiology Letters 202, 73–77.CrossRefGoogle Scholar
D'Ávila-levy, C. M., Souza, R. F., Gomes, R. C., Vermelho, A. B. and Branquinha, M. H. ( 2003). A metalloproteinase extracellularly released by Crithidia deanei. Canadian Journal of Microbiology 49, 625–632.CrossRefGoogle Scholar
D'Ávila-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 virulence factors of pathogenic trypanosomatids. Parasitology 130, 413–420.CrossRefGoogle Scholar
Etges, R. ( 1992). Identification of a surface metalloproteinase on 13 species of Leishmania isolated from humans, Crithidia fasciculata and Herpetomonas samuelpessoai. Acta Tropica 50, 205–217.CrossRefGoogle Scholar
Etges, R., Bouvier, J. and Bordier, C. ( 1986). The major surface protein of Leishmania promastigotes is a protease. The Journal of Biological Chemistry 261, 9099–9101.Google Scholar
Gorman, M. J. and Paskewitz, S. M. ( 2001). Serine proteases as mediators of mosquito immune responses. Insect Biochemistry and Molecular Biology 31, 257–262.CrossRefGoogle Scholar
Hajmová, M., Chang, K. P., Kolli, B. and Volf, P. ( 2004). Down-regulation of gp63 in Leishmania amazonensis reduces its early development in Lutzomyia longipalpis. Microbes and Infection 6, 646–649.CrossRefGoogle Scholar
Heussen, C. and Dowdle, E. B. ( 1980). Electrophoretic analysis of plasminogen activators in polyacrylamide gels containing sodium dodecyl sulphate and copolymerized substrates. Analytical Biochemistry 102, 196–202.CrossRefGoogle Scholar
Hughes, A. L. and Piontkivska, H. ( 2003). Molecular phylogenetics of Trypanosomatidae: contrasting results from 18S rRNA and protein phylogenies. Kinetoplastid Biology and Disease 2, 1–10.Google Scholar
Inverso, J. A., Medina-acosta, E., O'Connor, J., Russell, D. G. and Cross, G. A. ( 1993). Crithidia fasciculata contains a transcribed leishmanial surface proteinase (gp63) gene homologue. Molecular and Biochemical Parasitology 57, 47–54.CrossRefGoogle Scholar
Jansen, A. M., Carreira, J. C. and Deane, M. P. ( 1988). Infection of a mammal by monogenetic insect trypanosomatids (Kinetoplastida, Trypanosomatidae). Memórias do Instituto Oswaldo Cruz 83, 271–272.CrossRefGoogle Scholar
Jaffe, C. L. and Dwyer, D. M. ( 2003). Extracellular release of the surface metalloprotease gp63, from Leishmania and insect trypanosomatids. Parasitology Research 91, 229–237.CrossRefGoogle Scholar
Jones, B. L., Fontanini, D., Jarvinen, M. and Pekkarinen, A. ( 1998). Simplified endoproteinase assays using gelatin or azogelatin. Analytical Biochemistry 263, 214–220.CrossRefGoogle Scholar
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.CrossRefGoogle Scholar
Lacount, D. J., Gruszynski, A. E., Grandgnett, P. M., Bangs, J. D. and Donelson, J. E. ( 2003). Expression and function of the Trypanosoma brucei major surface protease (GP63) genes. The Journal of Biological Chemistry 278, 24658–24664.CrossRefGoogle Scholar
Laemmli, U. K. ( 1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, London 227, 680–685.CrossRefGoogle Scholar
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, 264–275.Google Scholar
MacDonald, M. H., Morrison, C. J. and McMaster, W. R. ( 1995). Analysis of the active site and activation mechanism of the Leishmania surface metalloproteinase GP63. Biochimica et Biophysica Acta 1253, 199–207.CrossRefGoogle Scholar
McGhee, R. B. and Cosgrove, W. B. ( 1980). Biology and physiology of the lower trypanosomatids. Microbiology Reviews 44, 140–173.Google Scholar
McGwire, B. S., O'Connell, W. A., Chang, K. P. and Engman, D. M. ( 2002). Extracellular release of the glycosylphosphatidylinositol (GPI)-linked Leishmania surface metalloprotease, gp63, is independent of GPI phospholipolysis. The Journal of Biological Chemistry 277, 8802–8809.CrossRefGoogle 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 metaloprotease gp63. Infection and Immunity 71, 1008–1010.CrossRefGoogle 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 Scholar
Medina-Acosta, E., Karess, R. E., Schwarz, H. and Russell, D. G. ( 1989). The promastigote surface protease (gp63) of Leishmania is expressed but differentially processed and localized in the amastigote stage. Molecular and Biochemical Parasitology 37, 263–274.CrossRefGoogle Scholar
Medina-Acosta, E., Beverley, S. M. and Russell, D. G. ( 1993). Evolution and expression of the Leishmania surface proteinase (gp63) gene locus. Infectious Agents and Disease 2, 25–34.Google Scholar
Melo, A. C. N., D'Avila-Levy, C. M., Branquinha, M. H. and Vermelho, A. B. ( 2002). Crithidia guilhermei: gelatin- and hemoglobin-degrading extracellular metalloproteinases. Experimental Parasitology 102, 150–156.CrossRefGoogle Scholar
Roitman, C., Roitman, I. and Azevedo, H. P. ( 1972). Growth of an insect trypanosomatid at 37 °C in a defined medium. Journal of Protozoology 19, 346–349.CrossRefGoogle Scholar
Santos, A. L. S., Ferreira, A., Franco, V. A., Alviano, C. S. and Soares, R. M. A. ( 1999). Characterization of proteinases in Herpetomonas anglusteri and Herpetomonas roitmani. Current Microbiology 39, 61–64.CrossRefGoogle Scholar
Santos, A. L. S., Abreu, C. M., Batista, L. M., Alviano, C. S. and Soares, R. M. A. ( 2001 a). Cell-associated and extracellular proteinases in Blastocrithidia culicis: influence of growth conditions. Current Microbiology 43, 100–106.Google Scholar
Santos, A. L. S., Batista, L. M., Abreu, C. M., Alviano, C. S., Angluster, J. and Soares, R. M. A. ( 2001 b). Developmentally regulated protein expression mediated by dimethylsulfoxide in Herpetomonas samuelpessoai. Current Microbiology 42, 111–116.Google Scholar
Santos, A. L. S., Abreu, C. M., Alviano, C. S. and Soares, R. M. A. ( 2002 a). Activation of the glycosylphosphatidylinositol membrane proteinase upon released from Herpetomonas samuelpessoai by phospholipase C. Current Microbiology 45, 293–298.Google Scholar
Santos, A. L. S., Rodrigues, M. L., Alviano, C. S. and Soares, R. M. A. ( 2002 b). Changes of sialomolecules during the dimethylsulfoxide-induced differentiation of Herpetomonas samuelpessoai. Parasitology Research 88, 951–955.Google Scholar
Santos, A. L. S., Souto-Padrón, T., Alviano, C. S., Lopes, A. H. C. S., Soares, R. M. A. and Meyer-Fernandes, J. R. ( 2002 c). Secreted phosphatase activity induced by dimethylsulfoxide in Herpetomonas samuelpessoai. Archives of Biochemistry and Biophysics 405, 191–198.Google Scholar
Santos, A. L. S., Rodrigues, M. L., Alviano, C. S., Angluster, J. and Soares, R. M. A. ( 2003). Herpetomonas samuelpessoai: dimethylsulfoxide-induced differentiation is influenced by proteinase expression. Current Microbiology 46, 11–17.CrossRefGoogle Scholar
Santos, A. L. S., Abreu, C. M., 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 Scholar
Schlein, Y., Schnur, L. F. and Jacobson, R. L. ( 1990). Released glycoconjugate of indigenous Leishmania major enhances survival of a foreign L. major in Phlebotomus papatasi. Transactions of the Royal Society of Tropical and Medicine Hygiene 84, 241–251.Google Scholar
Schneider, P. and Glaser, T. A. ( 1993). Characterization of a surface metalloprotease from Herpetomonas samuelpessoai and comparison with Leishmania major promastigote surface protease. Molecular and Biochemical Parasitology 58, 277–282.CrossRefGoogle Scholar
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.CrossRefGoogle Scholar
Stöcker, W. and Bode, W. ( 1995). Structural features of a superfamily of zinc-endopeptidases: the metzincins. Current Opinion in Structural Biology 5, 383–390.CrossRefGoogle Scholar
Tzinia, A. K. and Soteriadou, K. P. ( 1991). Substrate-dependent pH optima of gp63 purified from seven strains of Leishmania. Molecular and Biochemical Parasitology 47, 83–90.CrossRefGoogle Scholar
Vermelho, A. B., Almeida, F. V. S., Bronzato, L. S. and Branquinha, M. H. ( 2003). Extracellular metalloproteinases in Phytomonas serpens. Canadian Journal of Microbiology 49, 221–224.CrossRefGoogle Scholar
Weise, F., Stierhof, Y. D., Kuhn, C., Wiese, M. and Overath, P. ( 2000). Distribution of GPI-anchored proteins in the protozoan parasite Leishmania, based on an improved ultrastructural description using high-pressure frozen cells. The Journal of Cell Science 113, 4587–4603.Google Scholar
Yao, C., Leidal, K. G., Brittingham, A., Tarr, D. E., Donelson, J. E. and Wilson, M. E. ( 2003). Biosynthesis of the major surface protease GP63 of Leishmania chagasi. Molecular and Biochemical Parasitology 121, 119–128.Google Scholar
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