Hostname: page-component-cd9895bd7-gxg78 Total loading time: 0 Render date: 2024-12-23T07:49:23.014Z Has data issue: false hasContentIssue false
  • English
  • Français

Characterization of a Leishmania antigen associated with cytoplasmic vesicles resembling endosomal-like structure

Published online by Cambridge University Press:  06 April 2009

B. Yahiaoui ,
M. Loyens ,
A. Taibi ,
R. Schöneck ,
J. F. Dubremetz  and
M. A. Ouaissi
B. Yahiaoui
Affiliation:
Research Laboratory on Trypanosomatids, INSERM U 167, Institut Pasteur, Lille, France
M. Loyens
Affiliation:
Research Laboratory on Trypanosomatids, INSERM U 167, Institut Pasteur, Lille, France
A. Taibi
Affiliation:
Research Laboratory on Trypanosomatids, INSERM U 167, Institut Pasteur, Lille, France
R. Schöneck
Affiliation:
Research Laboratory on Trypanosomatids, INSERM U 167, Institut Pasteur, Lille, France
J. F. Dubremetz
Affiliation:
INSERM U42, Rue Jules Guesde, Domaine du Certia, Villeneuve d'ascq, Lille, France
M. A. Ouaissi
Affiliation:
Research Laboratory on Trypanosomatids, INSERM U 167, Institut Pasteur, Lille, France
Get access Rights & Permissions [Opens in a new window]

Summary

In the present study we have used antibodies to Leishmania major promastigote antigens which were eluted from a glutathione-agarose column (LmGbp) and could identify several parasite components among different Leishmania species by using immunoprecipitation and Western blot techniques. The results also showed that some of LmGbp are present among the molecules released into the culture medium. Moreover, immunofluorescence assays clearly demonstrated that LmGbp are expressed by intracellular amastigotes. The electron micrographs of thawed cryosections of L. major-infected cells revealed that the antigens were associated with the membrane of the phagocytic vacuole. Moreover, the Western blot technique allowed us to identify, using other Leishmania species extracts and anti-LmGbp antibodies, a major polypeptide of an apparent molecular mass of 66 kDa. Immunofluorescence studies suggested that the 66 kDa polypeptide is associated with intracytoplasmic vesicles. Cryosections of Leishmania promastigotes improved the fine structure preservation of the organelles and enabled a number of features to be seen, particularly the structures considered as vesicles, which appeared as a complex tubulo-vesicular structure resembling mammalian cell endosomes and Leishmania organelles previously named ‘megasomes’. Further studies using antibodies against the native 66 kDa protein will be needed to investigate the localization of the protein at the ultrastructural level and to follow its intracellular vesicular traffic.

Keywords

Leishmaniaimmunofluorescenceimmunolocalizationendosomal-like structure
Type
Research Article
Information
Parasitology , Volume 107 , Issue 5 , December 1993 , pp. 497 - 507
Copyright
Copyright © Cambridge University Press 1993

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. & Vickerman, K. (1975). Fusion of host cell secondary lysosomes with the parasitophorous vacuoles of Leishmania infected macrophages. Journal of Parasitology 22, 502–8.Google Scholar
Blocki, F. A., Schilevert, P. M. & Wackett, L. P. (1992). Rat liver protein linking chemical and immunological detoxification systems. Nature, London 360, 269–70.CrossRefGoogle ScholarPubMed
Coombs, G. H., Tetley, L., Moss, V. A. & Vickerman, K. (1986). Three-dimensional structure of theLeishmania amastigote as revealed by computer-aided reconstruction from serial section. Parasitology 92, 13–23.CrossRefGoogle Scholar
De Souza, W., De Carvalho, T. U. & Benchimol, M. (1978). Trypanosoma cruzi: ultrastructural, cytochemical and freeze-fracture studies of protein uptake. Experimental Parasitology 45, 101–15.CrossRefGoogle ScholarPubMed
Frommel, O. T., Button, L. L., Fujikura, Y. & McMaster, W. R. (1990). The major surface glycoprotein (GP63) is present in both life stages ofLeishmania. Molecular and Biochemical Parasitology 38, 25–32.CrossRefGoogle Scholar
Griffiths, G., Back, R. & Marsh, M. (1989). A quantitative analysis of the endocytic pathway in baby hamster kidney cells. Journal of Cell Biology 109, 2703–20.CrossRefGoogle ScholarPubMed
Gruenberg, J., Griffiths, G. & Howell, K. E. (1989). Characterization of the early endosome and putative endocytic carrier vesicles in vivo and with an assay of vesicle fusion in vitro. Journal of Cell Biology 108, 1301–16.CrossRefGoogle ScholarPubMed
Kweider, M., Lemesre, J. L., Darcy, F., Kusnierz, J. P., Capron, A. & Santoro, F. (1987). Infectivity ofLeishmania braziliensis promastigotes is dependent on the increasing expression of a 65,000 dalton surface antigen. Journal of Immunology 138, 299–305.CrossRefGoogle Scholar
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. Nature, London 227, 680–5.CrossRefGoogle ScholarPubMed
Le Ray, D., Afchain, D., Jadin, T., Capron, A., Yasarol, S. & Lanotte, G. (1973). Diagnostic immunoélectrophorétique de la leishmaniose viscérale à l'aide d'un extrait antigénique hydrosoluble de Leishmania donovani. Résultats préliminaires. Annales de la Société Belge Médecine Tropicale 53, 31–41.Google Scholar
Lowry, O. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951). Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry 193, 265–75.CrossRefGoogle ScholarPubMed
Medina-Acosta, E., Karess, R. E., Schwartz, H. & Russel, 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, 263–74.CrossRefGoogle ScholarPubMed
Morrissey, J. H. (1981). Silver stain for protein in polyacrylamide gels: modified procedure with enhanced uniform sensitivity. Annals of Biochemistry 117, 307–10.CrossRefGoogle ScholarPubMed
Olmsted, J. B. (1981). Affinity purification of antibodies from diazotised paper blots of heterogeneous protein samples. Journal of Biological Chemistry 250, 11955–7.CrossRefGoogle Scholar
Ouaissi, M. A., Taibi, A., Cornette, J., Velge, P., Marty, B., Loyens, M., Esteva, M., Rizvi, F. S. & Capron, A. (1990). Characterization of major surface and excretory-secretory immunogens ofTrypanosoma cruzi trypomastigotes and identification of potential protective antigen. Parasitology 100, 115–24.CrossRefGoogle Scholar
Ouaissi, M. A., Taibi, A., Loyens, M., Martin, U., Afchain, D., Maidana, C., Caudioti, C., Cornette, J., Martelleur, A., Velge, P., Marty, B. & Capron, A. (1991). Trypanosoma cruzi: a carbohydrate epitope defined by a monoclonal antibody as a possible marker of the acute phase of human Chagas' disease. American Journal of Tropical Medicine and Hygiene 45, 214–25.CrossRefGoogle ScholarPubMed
Plumas-Marty, B., Verwaerde, C., Loyens, M., Velge, P., Taibi, A., Cesbron, M. F., Capron, A. & Ouaissi, M. A. (1992). Trypanosoma cruzi glutathione binding proteins: immunogenicity during human and experimental chagas' disease. Parasitology 104, 87–98.CrossRefGoogle ScholarPubMed
Pupkis, M. F., Tetley, L. & Coombs, G. H. (1986). Leishmania mexicana: amastigote hydrolases in unusual lysosomes. Experimental Parasitology 62, 29–39.CrossRefGoogle ScholarPubMed
Russel, D. G. & Alexander, J. (1988). Effective immunization against cutaneous leishmaniasis with defined membrane antigens reconstituted into liposomes. Journal of Immunology 140, 1274–9.CrossRefGoogle Scholar
Sacks, D. L. (1989). Metacyclogenesis in Leishmania promastigotes. Experimental Parasitology 69, 100–3.CrossRefGoogle ScholarPubMed
Sacks, D. L. & Da Silva, R. M. (1987). The generation of infective stage Leishmania major promastigotes is associated with the cell-surface expression and release of a developmentally regulated glycolipid. Journal of Immunology 139, 3099–106.CrossRefGoogle ScholarPubMed
Sacks, D. L., Brodin, T. N. & Turco, S. J. (1990). Developmental modification of the lipophosphoglycan fromLeishmania major promastigotes during metacyclogenesis. Molecular and Biochemical Parasitology 42, 225–34.CrossRefGoogle ScholarPubMed
Schneider, P., Bordier, C. & Etges, R. (1993). Membrane proteins and enzymes ofLeishmania. Subcellular Biochemistry (in the Press).Google Scholar
Simons, P. C. & Vander Jagt, D. L. (1981). Purification of glutathione S-transferase by glutathione-affinity chromatography. Methods in Enzymology 77, 235–7.CrossRefGoogle ScholarPubMed
Towbin, H., Staehelin, T. & Gordon, J. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proceedings of the National Academy of Sciences, USA 76, 4350–4.CrossRefGoogle ScholarPubMed
Webster, P. & Fish, W. R. (1989). Endocytosis by African trypanosomes. II. Occurrence in different life-cycle stages and intracellular sorting. European Journal of Cell Biology 49, 303–10.Google ScholarPubMed