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Identification of a gene cluster for cell-surface genes of the SRS superfamily in Neospora caninum and characterization of the novel SRS9 gene

  • V. RISCO-CASTILLO (a1), V. MARUGÁN-HERNÁNDEZ (a1), A. FERNÁNDEZ-GARCÍA (a1), A. AGUADO-MARTÍNEZ (a1), E. JIMÉNEZ-RUIZ (a1), S. RODRÍGUEZ-MARCO (a1), G. ÁLVAREZ-GARCÍA (a1) and L. M. ORTEGA-MORA (a1)...

Summary

Here we present the detection of a gene cluster for Neospora caninum surface genes, similar to the Toxoplasma gondii SRS9 locus, and the cloning and characterization of the NcSRS9 gene. PCR genome walking, using NcBSR4 gene as a framework, allows the identification, upstream NcBSR4, of 2 sequences homologous to the SRS5 and the Ubiquinol-cytochrome C reductase genes and, downstream NcBSR4, of an ORF of 1191 bp coding for a 396-amino acid polypeptide with 59% similarity to the TgSRS9 antigen. A putative 39-residue signal peptide was found at the NH2-terminus followed by a hydrophilic region, and a potential site for a glycosylphosphatidylinositol anchor at the COOH-terminus. A recombinant NcSRS9 protein was produced and was recognized on a Western blot by a low proportion of sera from a panel of naturally infected cows and calves. In addition, Western blot analysis using polyclonal anti-rNcSRS9 revealed stage-specific expression of NcSRS9 in bradyzoites but not in tachyzoites, and immunohistochemistry on brain from a congenitally infected calf showed NcSRS9 recognition in bradyzoites contained in tissue cysts. However, bradyzoite-specific expression of NcSRS9 could not be proven by immunofluorescence on bradyzoites obtained in vitro and RT-PCR analysis showed no significant variations of NcSRS9 transcripts during in vitro tachyzoite-bradyzoite switch, probably due to incomplete maturity of in vitro bradyzoites. Initial characterization of NcSRS9 in this study may lead to further studies for a better understanding of N. caninum persistence.

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Corresponding author

*Corresponding author: Tel: +34 913944069. Fax: +34 913944098. E-mail: luis.ortega@vet.ucm.es

References

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Aguado-Martinez, A., Alvarez-Garcia, G., Fernandez-Garcia, A., Risco-Castillo, V., Arnaiz-Seco, I., Rebordosa-Trigueros, X., Navarro-Lozano, V. and 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, 182195.
Alvarez-Garcia, G., Pereira-Bueno, J., Gomez-Bautista, M. and Ortega-Mora, L. M. (2002). Pattern of recognition of Neospora caninum tachyzoite antigens by naturally infected pregnant cattle and aborted foetuses. Veterinary Parasitology 107, 1527.
Alvarez-Garcia, G., Pitarch, A., Zaballos, A., Fernandez-Garcia, A., Gil, C., Gomez-Bautista, M., Aguado-Martínez, A. and Ortega-Mora, L. M. (2007). The NcGRA7 gene encodes the immunodominant 17 kDa antigen of Neospora caninum. Parasitology 134, 4150.
Ajioka, J. W. and Soldati, D. (2007). Toxoplasma. Molecular and Cellular Biology. Horizon Bioscience. Norfolk, UK.
Anderson, M. L., Andrianarivo, A. G. and Conrad, P. A. (2000). Neosporosis in cattle. Animal Reproduction Science 60–61, 417431. doi: 10.1016/S0378-4320(00)00117-2.
Balaji, S., Babu, M. M., Iyer, L. M. and Aravind, L. (2005). Discovery of the principal specific transcription factors of Apicomplexa and their implication for the evolution of the AP2-integrase DNA binding domains. Nucleic Acids Research 33, 39944006. doi: 10.1093/nar/gki709.
Bjorkman, C. and Hemphill, A. (1998). Characterization of Neospora caninum iscom antigens using monoclonal antibodies. Parasite Immunology 20, 7380. doi: 10.1046/j.1365-3024.1998.00127.x
Cleary, M. D., Singh, U., Blader, I. J., Brewer, J. L. and Boothroyd, J. C. (2002). Toxoplasma gondii asexual development: identification of developmentally regulated genes and distinct patterns of gene expression. Eukaryotic Cell 1, 329340. doi: 10.1128/EC.1.3.329-340.2002.
Cross, G. A. (1990). Glycolipid anchoring of plasma membrane proteins. Annual Review of Cell Biology 6, 139. doi: 10.1146/annurev.cb.06.110190.000245.
Dubey, J. P. (2003). Review of Neospora caninum and neosporosis in animals. The Korean Journal of Parasitology 41, 116. doi: 10.3347/kjp.2003.41.1.1.
Dubey, J. P., Carpenter, J. L., Speer, C. A., Topper, M. J. and Uggla, A. (1988). Newly recognized fatal protozoan disease of dogs. Journal of the American Veterinary Medical Association 192, 12691285.
Dubey, J. P. and Schares, G. (2011). Neosporosis in animals – The last five years. Veterinary Parasitology (in the Press).
Fernandez-Garcia, A., Risco-Castillo, V., Zaballos, A., Varez-Garcia, G. and 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, 8997. doi: 10.1016/j.molbiopara.2005.08.019.
Gerber, L. D., Kodukula, K. and Udenfriend, S. (1992). Phosphatidylinositol glycan (PI-G) anchored membrane proteins. Amino acid requirements adjacent to the site of cleavage and PI-G attachment in the COOH-terminal signal peptide. The Journal of Biological Chemistry 267, 1216812173.
Gill, S. C. and von Hippel, P. H. (1989). Calculation of protein extinction coefficients from amino acid sequence data. Analytical Biochemistry 182, 319326.
Gondim, L. F., McAllister, M. M., Pitt, W. C. and Zemlicka, D. E. (2004). Coyotes (Canis latrans) are definitive hosts of Neospora caninum. International Journal for Parasitology 34, 159161. doi: 10.1016/j.ijpara.2004.01.001.
Hemphill, A. and Gottstein, B. (1996). Identification of a major surface protein on Neospora caninum tachyzoites. Parasitology Research 82, 497504.
Hemphill, A., Vonlaufen, N., Naguleswaran, A., Keller, N., Riesen, M., Guetg, N., Srinivasan, S. and Alaeddine, F. (2004). Tissue culture and explant approaches to studying and visualizing Neospora caninum and its interactions with the host cell. Microscopy and Microanalysis 10, 602620. doi: 10.1017/S1431927604040930.
Howe, D. K., Crawford, A. C., Lindsay, D. and Sibley, L. D. (1998). The p29 and p35 immunodominant antigens of Neospora caninum tachyzoites are homologous to the family of surface antigens of Toxoplasma gondii. Infection and Immunity 66, 53225328.
Howe, D. K. and Sibley, L. D. (1999). Comparison of the major antigens of Neospora caninum and Toxoplasma gondii. International Journal for Parasitology 29, 14891496. doi: 10.1016/S0020-7519(99)00099-5.
Jung, C., Lee, C. Y. and Grigg, M. E. (2004). The SRS superfamily of Toxoplasma surface proteins. International Journal for Parasitology 34, 285296. doi: 10.1016/j.ijpara.2003.12.004. doi: 10.1016/j.ijpara.2003.12.004.
Kang, S. W., Kweon, C. H., Lee, E. H., Choe, S. E., Jung, S. C. and Quyen, D. V. (2008). The differentiation of transcription between tachyzoites and bradyzoites of in vitro cultured Neospora caninum. Parasitology Research. doi: 10.1007/s00436-008-1082-5.
Kim, S. K. and Boothroyd, J. C. (2005). Stage-specific expression of surface antigens by Toxoplasma gondii as a mechanism to facilitate parasite persistence. Journal of Immunology 174, 80388048.
Kim, S. K., Karasov, A. and 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, 16261634. doi: 10.1128/IAI.01862-06.
King, J. S., Slapeta, J., Jenkins, D. J., Al-Qassab, S. E. and Ellis, J. T. (2010). Australian dingoes are definitive host of Neospora caninum. International Journal for Parasitology 40, 945950.
Knoll, L. J. and Boothroyd, J. C. (1998). Isolation of developmentally regulated genes from Toxoplasma gondii by a gene trap with the positive and negative selectable marker hypoxanthine-xanthine-guanine phosphoribosyltransferase. Molecular and Cellular Biology 18, 807814.
Krungkrai, J., Krungkrai, S. R., Suraveratum, N. and Prapunwattana, P. (1997). Mitochondrial ubiquinol-cytochrome c reductase and cytochrome c oxidase: chemotherapeutic targets in malarial parasites. Biochemistry and Molecular Biology International 42, 10071014.
Kyte, J. and Doolittle, R. F. (1982). A simple method for displaying the hydropathic character of a protein. Journal of Molecular Biology 157, 105132. doi: 10.1016/0022-2836(82)90515-0.
Lekutis, C., Ferguson, D. J., Grigg, M. E., Camps, M. and Boothroyd, J. C. (2001). Surface antigens of Toxoplasma gondii: variations on a theme. International Journal for Parasitology 31, 12851292. doi: 10.1016/S0020-7519(01)00261-2.
Lindsay, D. S. and Dubey, J. P. (1989). In vitro development of Neospora caninum (Protozoa: Apicomplexa) from dogs. The Journal of Parasitology 75, 163165.
Marugan-Hernandez, V., Alvarez-Garcia, G., Risco-Castillo, V., Regidor-Cerrillo, J. and Ortega-Mora, L. M. (2010). Identification of Neospora caninum proteins regulated during the differentiation process from tachyzoite to bradyzoite stage by DIGE. Proteomics 10, 17401750. doi: 10.1002/pmic.200900664.
McAllister, M. M., Parmley, S. F., Weiss, L. M., Welch, V. J. and 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. The Journal of Parasitology 82, 354355.
McFadden, D. C., Tomavo, S., Berry, E. A. and Boothroyd, J. C. (2000). Characterization of cytochrome b from Toxoplasma gondii and Q(o) domain mutations as a mechanism of atovaquone-resistance. Molecular and Biochemical Parasitology 108, 112. doi: 10.1016/S0166-6851(00)00184-5.
Morales-Sainz, L., Escobar-Ramirez, A., Cruz-Torres, V., Reyes-Prieto, A., Vazquez-Acevedo, M., Lara-Martinez, R., Jimenez-Garcia, L. F. and Gonzalez-Halphen, D. (2008). The polypeptides COX2A and COX2B are essential components of the mitochondrial cytochrome c oxidase of Toxoplasma gondii. Biochimica et Biophysica Acta 1777, 202210. doi: 10.1016/j.bbabio.2007.10.013.
Nishikawa, Y., Xuan, X., Nagasawa, H., Igarashi, I., Fujisaki, K., Otsuka, H. and Mikami, T. (2000). Monoclonal antibody inhibition of Neospora caninum tachyzoite invasion into host cells. International Journal for Parasitology 30, 5158. doi:10.1016/S0020-7519(99)00162-9
Pereira-Bueno, J., Quintanilla-Gozalo, A., Perez-Perez, V., Espi-Felgueroso, A., Alvarez, G., Collantes-Fernandez, E. and Ortega-Mora, L. M. (2003). Evaluation by different diagnostic techniques of bovine abortion associated with Neospora caninum in Spain. Veterinary Parasitology 111, 143152. doi: 10.1016/S0304-4017(02)00361-8.
Regidor-Cerrillo, J., Gomez-Bautista, M., Pereira-Bueno, J., Aduriz, G., Navarro-Lozano, V., Risco-Castillo, V., Fernandez-García, A., Pedraza-Diaz, S. and Ortega-Mora, L. M. (2008). Isolation and genetic characterization of Neospora caninum from asymptomatic calves in Spain. Parasitology 135, 16511659. doi:10.1017/S003118200800509X.
Risco-Castillo, V., Fernández-García, A. and 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, 466470. DOI: 10.1043/0022-3395
Risco-Castillo, V., Fernández-García, A., Zaballos, A., Aguado-Martínez, A., Hemphill, A., Rodríguez-Bertos, A., Álvarez-García, G. and Ortega-Mora, L. M. (2007). Molecular characterisation of BSR4, a novel bradyzoite-specific gene from Neospora caninum. International Journal for Parasitology 37, 887896. doi: 10.1016/j.ijpara.2007.02.003.
Vonlaufen, N., Guetg, N., Naguleswaran, A., Muller, N., Bjorkman, C., Schares, G., von Blumroeder, D., Ellis, J. and Hemphill, A. (2004). In vitro induction of Neospora caninum bradyzoites in vero cells reveals differential antigen expression, localization, and host-cell recognition of tachyzoites and bradyzoites. Infection and Immunity 72, 576583. doi: 10.1128/IAI.72.1.576–583.2004.
Yang, S. and Parmley, S. F. (1997). Toxoplasma gondii expresses two distinct lactate dehydrogenase homologous genes during its life cycle in intermediate hosts. Gene 184, 112. doi: 10.1016/S0378-1119(96)00566-5.
Zhao, J. C., Zhao, Z. D., Wang, W. and Gao, X. M. (2005). Prokaryotic expression, refolding, and purification of fragment 450–650 of the spike protein of SARS-coronavirus. Protein Expression and Purification 39, 169174. doi: 10.1016/j.pep.2004.10.004.

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