Acta Parasitologica

, Volume 55, Issue 4, pp 304–312 | Cite as

Characterisation of NcGRA7 and NcSAG4 proteins: Immunolocalisation and their role in the host cell invasion by Neospora caninum tachyzoites

  • Adriana Aguado-Martínez
  • Gema Álvarez-García
  • Gereon Schares
  • Verónica Risco-Castillo
  • Aurora Fernández-García
  • Virginia Marugán-Hernández
  • Luis M. Ortega-Mora
Article

Abstract

Neospora caninum negatively impacts bovine reproductive performance around the world. Addressing this problem requires a greater understanding of the parasite’s molecular biology. In this study, monoclonal antibodies against recombinant proteins were successfully developed and employed to characterise two different proteins of N. caninum: the acute phase-associated NcGRA7 and the chronic phase-associated NcSAG4. Immunofluorescence with the anti-rNcGRA7 monoclonal antibody suggested that NcGRA7 trafficks from tachyzoite dense granules to the matrix of the parasitophorous vacuole and parasite’s surroundings. Furthermore, NcGRA7 is also expressed in the bradyzoite stage and localised on the matrix of bradyzoite-positive vacuoles. NcGRA7 appears to be partially involved in the tachyzoite-invasion mechanisms, as an anti-rNcGRA7 monoclonal antibody partially inhibited in vitro tachyzoite-invasion. A monoclonal antibody specific for NcSAG4 confirmed this protein’s bradyzoitespecific expression both by western blot and immunofluorescence. However, some bradyzoite-positive vacuoles only weakly expressed NcSAG4, if it was expressed at all. The specificity of the anti-rNcSAG4 monoclonal antibody was confirmed by the recognition of the NcSAG4 in the membrane surface of Nc-1SAG4c transgenic tachyzoites, which constitutively expresses NcSAG4. Blocking NcSAG4 of Nc-1SAG4c tachyzoites with the monoclonal antibody did not affect host cell invasion. However, its implication on the host cell adhesion or host immune evasion should not be discarded.

Keywords

NcGRA7 NcSAG4 immunolocalisation invasion Neospora caninum tachyzoite bradyzoite monoclonal antibody 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Aguado-Martínez A., Álvarez-García G., Fernández-García A., Risco-Castillo V., Arnaiz-Seco I., Rebordosa-Trigueros X., Navarro-Lozano V., Ortega-Mora L.M. 2008. Usefulness of rNcGRA7- and rNcSAG4-based ELISA tests for distinguishing primo-infection, recrudescence, and chronic bovine neosporosis. Veterinary Parasitology, 157, 182–195. DOI: 10.1016/j.vetpar.2008.08.002.CrossRefPubMedGoogle Scholar
  2. Aguado-Martínez A., Álvarez-García G., Fernández-García A., Risco-Castillo V., Marugán-Hernández V., Ortega-Mora L.M. 2009a. Failure of a vaccine using immunogenic recombinant proteins rNcSAG4 and rNcGRA7 against neosporosis in mice. Vaccine, 27, 7331–7338. DOI: 10.1016/j.vaccine.2009.09.050.CrossRefPubMedGoogle Scholar
  3. Aguado-Martínez A., Ortega-Mora L.M., Álvarez-García G., Rodríguez-Marco S., Risco-Castillo V., Marugán-Hernández V., Fernández-García A. 2009b. Stage-specific expression of NcSAG4 as a marker of chronic Neospora caninum infection in a mouse model. Parasitology, 136, 757–764. DOI: 10.1017/S0031182009006076.CrossRefPubMedGoogle Scholar
  4. Álvarez-García G., Pitarch A., Zaballos A., Fernández-García A., Gil C., Gómez-Bautista M., Aguado-Martínez A., Ortega-Mora L.M. 2007. The NcGRA7 gene encodes the immunodominant 17 kDa antigen of Neospora caninum. Parasitology, 134, 41–50. DOI: 10.1017/S0031182006001284.CrossRefPubMedGoogle Scholar
  5. Augustine P.C., Jenkins M.C., Dubey J.P. 1999. Effect of polyclonal antisera developed against dense granule-associated Neospora caninum proteins on cell invasion and development in vitro by N. caninum tachyzoites. Parasitology, 119, 441–445. DOI: 10.1017/S003118209900493X.CrossRefPubMedGoogle Scholar
  6. Barber J., Trees A.J., Owen M., Tennant B. 1993. Isolation of Neospora caninum from a British dog. Veterinary Record, 133, 531–532.CrossRefPubMedGoogle Scholar
  7. Bohne W., Gross U., Ferguson D.J., Heesemann J. 1995. Cloning and characterization of a bradyzoite-specifically expressed gene (hsp30/bag1) of Toxoplasma gondii, related to genes encoding small heat-shock proteins of plants. Molecular Microbiology, 16, 1221–1230.CrossRefPubMedGoogle Scholar
  8. Bonhomme A., Maine G.T., Beorchia A., Burlet H., Aubert D., Villena I., Hunt J., Chovan L., Howard L., Brojanac S., Sheu M., Tyner J., Pluot M., Pinon J.M. 1998. Quantitative immunolocalization of a P29 protein (GRA7), a new antigen of Toxoplasma gondii. Journal of Histochemistry and Cytochemistry, 46, 1411–1422.PubMedGoogle Scholar
  9. Boothroyd J.C., Black M., Bonnefoy S., Hehl A., Knoll L.J., Manger I.D., Ortega-Barria E., Tomavo S. 1997. Genetic and biochemical analysis of development in Toxoplasma gondii. Philosophical Transaction of the Royal Society of London, Series B, Biological Sciences, 352, 1347–1354. DOI: 10.1098/rstb.1997.0119.Google Scholar
  10. Braun L., Travier L., Kieffer S., Musset K., Garin J., Mercier C., Cesbron-Delauw M.F. 2008. Purification of Toxoplasma dense granule proteins reveals that they are in complexes throughout the secretory pathway. Molecular and Biochemical Parasitology, 157, 13–21. DOI: 10.1016/j.molbiopara.2007.09.002.CrossRefPubMedGoogle Scholar
  11. Cesbron-Delauw M.F. 1994. Dense-granule organelles of Toxoplasma gondii: their role in the host-parasite relationship. Parasitology Today, 10, 293–296. DOI: 10.1016/0169-4758(94)90078-7.CrossRefPubMedGoogle Scholar
  12. Cesbron-Delauw M.F., Lecordier L., Mercier C. 1996. Role of secretory dense granule organelles in the pathogenesis of toxoplasmosis. Current Topics in Microbiology and Immunology, 219, 59–65.PubMedGoogle Scholar
  13. Cho J.H., Chung W.S., Song K.J., Na B.K., Kang S.W., Song C.Y., Kim T.S. 2005. Protective efficacy of vaccination with Neospora caninum multiple recombinant antigens against experimental Neospora caninum infection. Korean Journal of Parasitology, 43, 19–25.CrossRefPubMedGoogle Scholar
  14. Di Cristina M., Del Porto P., Buffolano W., Beghetto E., Spadoni A., Guglietta S., Piccolella E., Felici F., Gargano N. 2004. The Toxoplasma gondii bradyzoite antigens BAG1 and MAG1 induce early humoral and cell-mediated immune responses upon human infection. Microbes and Infection, 6, 164–171. DOI: 10.1016/j.micinf.2003.11.009.CrossRefPubMedGoogle Scholar
  15. Dubey J.P., Schares G. 2006. Diagnosis of bovine neosporosis. Veterinary Parasitology, 140, 1–34. DOI: 10.1016/j.vetpar.2006.03.035.CrossRefPubMedGoogle Scholar
  16. Dubey J.P., Schares G., Ortega-Mora L.M. 2007. Epidemiology and control of neosporosis and Neospora caninum 18. Clinical Microbiology Reviews, 20, 323–367. DOI: 10.1128/CMR.00031-06.CrossRefPubMedGoogle Scholar
  17. Fernández-García A., Risco-Castillo V., Zaballos A., Álvarez-García G., Ortega-Mora L.M. 2006. Identification and molecular cloning of the Neospora caninum SAG4 gene specifically expressed at bradyzoite stage. Molecular and Biochemical Parasitology, 146, 89–97. DOI: 10.1016/j.molbiopara.2005.08.019.CrossRefPubMedGoogle Scholar
  18. Fischer H.G., Stachelhaus S., Sahm M., Meyer H.E., Reichmann G. 1998. GRA7, an excretory 29 kDa Toxoplasma gondii dense granule antigen released by infected host cells. Molecular and Biochemical Parasitology, 91, 251–262. DOI: 10.1016/S0166-6851(97)00227-2.CrossRefPubMedGoogle Scholar
  19. Fuchs N., Sonda S., Gottstein B., Hemphill A. 1998. Differential expression of cell surface- and dense granule-associated Neospora caninum proteins in tachyzoites and bradyzoites. Journal of Parasitology, 84, 753–758. DOI: 10.2307/3284583.PubMedGoogle Scholar
  20. Ghalmi F., China B., Kaidi R., Losson B. 2009. Evaluation of a SRS2 sandwich commercial enzyme-linked immunosorbent assay for the detection of anti-Neospora caninum antibodies in bovine and canine sera. Journal of Veterinary Diagnostic Investigation, 21, 108–111.PubMedGoogle Scholar
  21. Hemphill A., Gottstein B., Kaufmann H. 1996. Adhesion and invasion of bovine endothelial cells by Neospora caninum. Parasitology, 112, 183–197. DOI: 10.1017/S0031182000084754.CrossRefPubMedGoogle Scholar
  22. Hemphill A., Gajendran N., Sonda S., Fuchs N., Gottstein B., Hentrich B., Jenkins M. 1998. Identification and characterisation of a dense granule-associated protein in Neospora caninum tachyzoites. International Journal for Parasitology, 28, 429–438. DOI: 10.1016/S0020-7519(97)00193-8.CrossRefPubMedGoogle Scholar
  23. Hemphill A. 1999. The host-parasite relationship in neosporosis. Advances in Parasitology, 43, 47–104. DOI: 10.1016/S0065- 308X.CrossRefPubMedGoogle Scholar
  24. Jenkins M.C., Wouda W., Dubey J.P. 1997. Serological response over time to recombinant Neospora caninum antigens in cattle after a neosporosis-induced abortion. Clinical and Diagnostic Laboratory Immunology, 4, 270–274.PubMedGoogle Scholar
  25. Jongert E., De Craeye S., Dewit J., Huygen K. 2007. GRA7 provides protective immunity in cocktail DNA vaccines against Toxoplasma gondii. Parasite Immunology, 29, 445–453. DOI: 10.1111/j.1365-3024.2007.00961.x.CrossRefPubMedGoogle Scholar
  26. Kim S.K., Boothroyd J.C. 2005. Stage-specific expression of surface antigens by Toxoplasma gondii as a mechanism to facilitate parasite persistence. Journal of Immunology, 174, 8038–8048.Google Scholar
  27. Kim S.K., Karasov A., Boothroyd J.C. 2007. Bradyzoite-specific surface antigen SRS9 plays a role in maintaining Toxoplasma gondii persistence in the brain and in host control of parasite replication in the intestine. Infection and Immunity, 75, 1626–1634. DOI: 10.1128/IAI.01862-06.CrossRefPubMedGoogle Scholar
  28. Lally N., Jenkins M., Liddell S., Dubey J.P. 1997. A dense granule protein (NCDG1) gene from Neospora caninum. Molecular and Biochemical Parasitology, 87, 239–243. DOI: 10.1016/S0166-6851(97)00070-4.CrossRefPubMedGoogle Scholar
  29. Lally N.C., Jenkins M.C., Dubey J.P. 1996. Evaluation of two Neospora caninum recombinant antigens for use in an enzyme- linked immunosorbent assay for the diagnosis of bovine neosporosis. Clinical and Diagnostic Laboratory Immunology, 3, 275–279.PubMedGoogle Scholar
  30. Marugán-Hernández V., Ortega-Mora L.M., Aguado-Martínez A., Álvarez-García G., submitted. Genetic manipulation of Neospora caninum tachyzoites to obtain a low virulence strain.Google Scholar
  31. McAllister M.M., Parmley S.F., Weiss L.M., Welch V.J., McGuire A.M. 1996. An immunohistochemical method for detecting bradyzoite antigen (BAG5) in Toxoplasma gondii-infected tissues cross-reacts with a Neospora caninum bradyzoite antigen. Journal of Parasitology, 82, 354–355.CrossRefPubMedGoogle Scholar
  32. Neudeck A., Stachelhaus S., Nischik N., Striepen B., Reichmann G., Fischer H.G. 2002. Expression variance, biochemical and immunological properties of Toxoplasma gondii dense granule protein GRA7. Microbes and Infection, 4, 581–590. DOI: 10.1016/S1286-4579(02)01576-9.CrossRefPubMedGoogle Scholar
  33. Pérez-Zaballos F.J., Ortega-Mora L.M., Álvarez-García G., Collantes-Fernández E., Navarro-Lozano V., García-Villada L., Costas E. 2005. Adaptation of Neospora caninum isolates to cell-culture changes: an argument in favor of its clonal population structure. Journal of Parasitology, 91, 507–510.CrossRefPubMedGoogle Scholar
  34. Reichel M.P., Ellis J.T. 2006. If control of Neospora caninum infection is technically feasible does it make economic sense? Veterinary Parasitology, 142, 23–34. DOI: 10.1016/j.vetpar.2006. 06.027.CrossRefPubMedGoogle Scholar
  35. Reichel M.P., Ellis J.T. 2009. Neospora caninum — how close are we to development of an efficacious vaccine that prevents abortion in cattle? International Journal for Parasitology, 39, 1173–1187. DOI: 10.1016/j.ijpara.2009.05.007.CrossRefPubMedGoogle Scholar
  36. Risco-Castillo V., Fernández-García A., Ortega-Mora L.M. 2004. Comparative analysis of stress agents in a simplified in vitro system of Neospora caninum bradyzoite production. Journal of Parasitology, 90, 466–470.CrossRefPubMedGoogle Scholar
  37. Risco-Castillo V., Fernández-García A., Zaballos A., Aguado-Martínez A., Hemphill A., Rodríguez-Bertos A., Álvarez-García G., Ortega-Mora L.M. 2007. Molecular characterisation of BSR4, a novel bradyzoite-specific gene from Neospora caninum. International Journal for Parasitology, 37, 887–896. DOI: 10.1016/j.ijpara.2007.02.003.CrossRefPubMedGoogle Scholar
  38. Schares G., Dubremetz J.F., Dubey J.P., Barwald A., Loyens A., Conraths F.J. 1999. Neospora caninum: identification of 19-, 38-, and 40-kDa surface antigens and a 33-kDa dense granule antigen using monoclonal antibodies. Experimental Parasitology, 92, 109–119. DOI: 10.1006/expr.1999.4403.CrossRefPubMedGoogle Scholar
  39. Schwarz J.A., Fouts A.E., Cummings C.A., Ferguson D.J., Boothroyd J.C. 2005. A novel rhoptry protein in Toxoplasma gondii bradyzoites and merozoites. Molecular and Biochemical Parasitology, 144, 159–166. DOI: 10.1016/j.molbiopara.2005. 08.011.CrossRefPubMedGoogle Scholar
  40. Silva D.A., Lobato J., Mineo T.W., Mineo J.R. 2007. Evaluation of serological tests for the diagnosis of Neospora caninum infection in dogs: Optimization of cut off titers and inhibition studies of cross-reactivity with Toxoplasma gondii. Veterinary Parasitology, 143, 234–244. DOI: 10.1016/j.vetpar.2006. 08.028.CrossRefPubMedGoogle Scholar
  41. Vaitukaitis J.L. 1981. Production of antisera with small doses of immunogen: multiple intradermal injections. Methods in Enzymology, 73, 46–52.CrossRefPubMedGoogle Scholar
  42. Vonlaufen N., Guetg N., Naguleswaran A., Müller N., Björkman C., Schares G., von Blumroeder D., Ellis J., Hemphill A. 2004. In vitro induction of Neospora caninum bradyzoites in vero cells reveals differential antigen expression, localization, and hostcell recognition of tachyzoites and bradyzoites. Infection and Immunity, 72, 576–583.CrossRefPubMedGoogle Scholar
  43. Weiss L.M., Ma Y.F., Halonen S., McAllister M.M., Zhang Y.W. 1999. The in vitro development of Neospora caninum bradyzoites. International Journal for Parasitology, 29, 1713–1723. DOI: 10.1016/S0020-7519(99)00130-7.CrossRefPubMedGoogle Scholar

Copyright information

© © Versita Warsaw and Springer-Verlag Wien 2010

Authors and Affiliations

  • Adriana Aguado-Martínez
    • 1
  • Gema Álvarez-García
    • 1
  • Gereon Schares
    • 2
  • Verónica Risco-Castillo
    • 3
  • Aurora Fernández-García
    • 4
  • Virginia Marugán-Hernández
    • 1
  • Luis M. Ortega-Mora
    • 1
  1. 1.Grupo SALUVET, Departamento de Sanidad Animal, Facultad de VeterinariaUniversidad Complutense de MadridMadridSpain
  2. 2.Friedrich-Loeffler-Institut, Federal Research for Animal HealthInstitute of ParasitologyWusterhausenGermany
  3. 3.Institut National de la Santé et de la Recherche Médicale (INSERM)Unité Mixte de Recherche S945ParisFrance
  4. 4.Departamento de Biología y Variabilidad del VIH, CNMInstituto de Salud Carlos IIIMajadahonda, MadridSpain

Personalised recommendations