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

• Autorzy: Aguado-Martinez A , Alvarez-Garcia G. , Schares G. , Risco-Castillo V. , Fernandez-Garcia A. , Marugan-Hernandez V. , Ortega-Mora L.M.
• Języki publikacji: EN

Abstrakty

EN

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.

Słowa kluczowe

EN

NcGRA7 protein; NcSAG4 protein; immunolocalization; host cell; invasion; Neospora caninum; tachyzoite; bradyzoite; parasite; molecular biology; monoclonal antibody; recombinant protein; immunofluorescence; immunoblot

• Wydawca: -
• Czasopismo: Acta Parasitologica
• Rocznik: 2010
• Tom: 55
• Numer: 4
• Opis fizyczny: p.304-312,fig.,ref.

Twórcy

autor

Aguado-Martinez A

• Universidad Complutense de Madrid, Ciudad Universitaria s/n, 28040-Madrid, Spain

autor

Alvarez-Garcia G.

autor

Schares G.

autor

Risco-Castillo V.

autor

Fernandez-Garcia A.

autor

Marugan-Hernandez V.

autor

Ortega-Mora L.M.

Bibliografia

• 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.
• 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.
• 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.
• 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.
• 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.
• Barber J., Trees A.J., Owen M., Tennant B. 1993. Isolation of Neospora caninum from a British dog. Veterinary Record, 133, 531–532.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• Dubey J.P., Schares G. 2006. Diagnosis of bovine neosporosis. Veterinary Parasitology, 140, 1–34. DOI: 10.1016/j.vetpar.2006.03.035.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• Hemphill A. 1999. The host-parasite relationship in neosporosis. Advances in Parasitology, 43, 47–104. DOI: 10.1016/S0065- 308X.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• Vaitukaitis J.L. 1981. Production of antisera with small doses of immunogen: multiple intradermal injections. Methods in Enzymology, 73, 46–52.
• 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.
• 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.