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Molecular characterizations of three distinct Babesia gibsoni rhoptry-associated protein-1s (RAP-1s)

  • M. A. TERKAWI (a1), A. AMORNTHEP (a2), H. OOKA (a1), G. ABOGE (a1), H. JIA (a1), Y.-K. GOO (a1), B. NELSON (a1), J. YAMAGISHI (a1), Y. NISHIKAWA (a1), I. IGARASHI (a1), S.-I. KAWAZU (a1), K. FUJISAKI (a3) and X. XUAN (a1)...

Summary

Three cDNAs encoding rhoptry-associated protein 1 (RAP-1) homologues were found in the Babesia gibsoni EST database. Based on similarities to BgRAP-1a, which was identified previously by serological screening of a cDNA merozoite library, the two new genes were designated BgRAP-1b (33·7%) and BgRAP-1c (57%). Mice antiserum raised against each recombinant protein reacted specifically with B. gibsoni parasites as determined by Western blotting, which showed native molecular sizes of the BgRAP-1a (51 kDa), BgRAP-1b (53 kDa) and BgRAP-1c (47 kDa) consistent with predictable molecular weights. Immunofluoresence using these antibodies revealed localization of all BgRAP-1s within the matrix of merozoites; however, BgRAP-1a appeared to diverge from the other two when it was found secreted into the cytoplasm of infected erythrocytes. Apical localization of all 3 BgRAP-1s during the extracellular stage of the parasite combined with their ability to bind a canine erythrocyte membrane fraction was suggestive of a role for these proteins in erythrocyte attachment. Lastly, the ability of these recombinant proteins to be used as diagnostic reagents was tested by ELISA and the sensitivities of BgRAP-1a and BgRAP-1c were found increased through N-terminal truncation. Taken together, our data suggest divergent roles for the 3 BgRAP-1s in the merozoite stage of B. gibsoni.

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Corresponding author

*Corresponding author: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan. Tel: +81 155 49 5648. Fax: +81 155 49 5643. E-mail: gen@obihiro.ac.jp

References

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Blackman, M. J. and Bannister, L. H. (2001). Apical organelles of Apicomplexa: biology and isolation by subcellular fractionation. Molecular and Biochemical Parasitology 117, 1125.
Boonchit, S., Xuan, X., Yokoyama, N., Goff, W. L., Wagner, G. and Igarashi, I. (2002). Evaluation of an enzyme-linked immunosorbent assay with recombinant rhoptry-associated protein 1 antigen against Babesia bovis for the detection of specific antibodies in cattle. Journal of Clinical Microbiology 40, 37713775.
Boonchit, S., Xuan, X., Yokoyama, N., Goff, W. L., Waghela, S. D., Wagner, G. and Igarashi, I. (2004). Improved enzyme-linked immunosorbent assay using C-terminal truncated recombinant antigens of Babesia bovis rhoptry-associated protein-1 for detection of specific antibodies. Journal of Clinical Microbiology 42, 16011604.
Boonchit, S., Alhassan, A., Chan, B., Xuan, X., Yokoyama, N., Ooshiro, M., Goff, W. L., Waghela, S. D., Wagner, G. and Igarashi, I. (2006). Expression of C-terminal truncated and full-length Babesia bigemina rhoptry-associated protein 1 and their potential use in enzyme-linked immunosorbent assay. Veterinary Parasitology 137, 2835.
Boozer, A. L. and Macintire, D. K. (2003). Canine babesiosis. Veterinary Clinics Small Animal Practice 33, 885904.
Brown, W. C. and Palmer, G. H. (1999). Designing blood-stage vaccines against Babesia bovis and B. bigemina. Parasitology Today 15, 275281.
Brown, W. C., Norimine, J., Goff, W. L., Suarez, C. E. and McElwain, T. F. (2006). Prospects for recombinant vaccines against Babesia bovis and related parasites. Parasite Immunology 28, 315327.
Calvo, M., Guzmán, F., Perez, E., Segura, C. H., Molano, A. and Patarroyo, M. E. (1991). Specific interactions of synthetic peptides derived from Plasmodium falciparum merozoite proteins with human red blood cells. Peptide Research 4, 324332.
Curtidor, H., Ocampo, M., Tovar, D., López, R., García, J., Valbuena, J., Vera, R., Suárez, J., Rodríguez, L. E., Puentes, A., Guzmán, F., Torres, E. and Patarroyo, M. E. (2004). Specific erythrocyte binding capacity and biological activity of P. falciparum-derived rhoptry-associated protein 1 peptides. Vaccine 8, 10541062.
Dalrymple, B. P. (1993). Molecular variation and diversity in candidate vaccine antigens from Babesia. Acta Tropica 53, 227238.
Dalrymple, B. P., Casu, R. E., Peters, J. M., Dimmock, C. M., Gale, K. R., Boese, R. and Wright, I. G. (1993). Characterization of a family of multi-copy genes encoding rhoptry protein homologues in Babesia bovis, B. ovis and B. canis. Molecular and Biochemical Parasitology 57, 181192.
Fish, L., Leibovich, B., Krigel, Y., McElwain, T. and Shkap, V. (2008). Vaccination of cattle against B. bovis infection with live attenuated parasites and non-viable immunogens. Vaccine 26, 2933.
Fukumoto, S., Xuan, X., Nishikawa, Y., Inoue, N., Igarashi, I., Nagasawa, H., Fujisaki, K. and Mikami, T. (2001). Identification and expression of a 50-kilodalton surface antigen of Babesia gibsoni and evaluation of its diagnostic potential in an enzyme-linked immunosorbent assay. Journal of Clinical Microbiology 39, 26032609.
Gaffar, F. R., Yatsuda, A. P., Franssen, F. F. and de Vries, E. (2004 a). Erythrocyte invasion by Babesia bovis merozoites is inhibited by polyclonal antisera directed against peptides derived from a homologue of Plasmodium falciparum apical membrane antigen 1. Infection and Immunity 72, 29472955.
Gaffar, F. R., Yatsuda, A. P., Franssen, F. F. and de Vries, E. (2004 b). A Babesia bovis merozoite protein with a domain architecture highly similar to the thrombospondin-related anonymous protein (TRAP) present in Plasmodium sporozoites. Molecular and Biochemical Parasitology 136, 2534.
Goff, W. L., Davis, W. C., Palmer, G. H., McElwain, T. F., Johnson, W. C. and Bailey, J. F. (1988). Identification of Babesia bovis merozoite surface antigens by using immune bovine sera and monoclonal antibodies. Infection and Immunity 56, 23632368.
Goo, Y. K., Jia, H., Aboge, G. O., Terkawi, M. A., Kuriki, K., Nakamura, C., Kumagai, A., Zhou, J., Lee, E. G., Nishikawa, Y., Igarashi, I., Fujisaki, K. and Xuan, X. (2008). Babesia gibsoni: Serodiagnosis of infection in dogs by an enzyme-linked immunosorbent assay with recombinant BgTRAP. Experimental Parasitology 118, 555560.
Homer, M. J., Aguilar-Delfin, I., Telford, S. R., Krause, P. J. and Persing, D. H. (2000). Babesiosis. Clinical Microbiology Review 13, 451469.
Howard, R. F., Jacobson, K. C., Rickel, E. and Thurman, J. (1998). Analysis of inhibitory epitopes in the Plasmodium falciparum rhoptry protein RAP-1 including identification of a second inhibitory epitope. Infection and Immunity 66, 380386.
Ishimine, T., Makimura, S., Kitazawa, S., Tamura, S. and Suzuki, N. (1978). Pathophysiological findings on blood of beagles experimentally infected with B. gibsoni. The Japanese Journal of Tropical Medicine Hygiene 6, 1526.
Johnson, W. C., Perryman, L. E. and Goff, W. L. (1997). Babesia bovis: identification of immunodominant merozoite surface proteins in soluble culture-derived exoantigen. Parasitology Research 83, 776780.
Kaneko, O. (2007). Erythrocyte invasion: vocabulary and grammar of the Plasmodium rhoptry. Parasitology International 56, 255262.
Kato, S., Ohtoko, K., Ohtake, H. and Kimura, T. (2005). Vector-capping: a simple method for preparing a high-quality full-length cDNA library. DNA Research 12, 5362.
Kumar, S., Yokoyama, Y., Kim, J. Y., Huang, X., Inoue, N., Xuan, X., Igarashi, I. and Sugimoto, C. (2004). Expression of Babesia equi EMA-1 and EMA-2 during merozoite developmental stages in erythrocyte and their interaction with erythrocytic membrane skeleton. Molecular and Biochemical Parasitology 133, 221227.
Kuttler, K. L. (1988). Worldwide impact of babesiosis. In Babesiosis of Domestic Animals (ed. Ristic, M.), pp. 122. CRC Press Inc., Boca Raton. FL, USA.
López, R., Valbuena, J., Curtidor, H., Puentes, A., Rodríguez, L. E., García, J., Suárez, J., Vera, R., Ocampo, M., Trujillo, M., Ramirez, L. E. and Patarroyo, M. E. (2004). Plasmodium falciparum: red blood cell binding studies using peptides derived from rhoptry-associated protein 2 (RAP2). Biochimie 86, 16.
Machado, R. Z., McElwain, T. F., Pancracio, H. P., Freschi, C. R. and Palmer, G. H. (1999). Babesia bigemina: immunization with purified rhoptries induces protection against acute parasitemia. Experimental Parasitology 93, 105108.
Machado, R. Z., Palmer, G. H. and McElwain, T. F. (1997). Vaccination of cattle with Babesia bigemina rhoptry proteins. In Proceedings from the Society for Tropical Veterinary Medicine 4th Biennial Meeting, Montpellier, France, p. 46.
Macintire, D. K., Boudreaux, M. K., West, G. D., Bourne, C., Wright, J. C. and Conrad, P. A. (2002). Babesia gibsoni infection among dogs in the southeastern United States. Journal of the American Veterinary Medical Association 220, 325329.
McElwain, T. F., Perryman, L. E., Davis, W. C. and McGuire, T. C. (1987). Antibodies define multiple proteins with epitopes exposed on the surface of live Babesia bigemina merozoites. Journal of Immunology 138, 22982304.
McElwain, T. F., Perryman, L. E., Musoke, A. J. and McGuire, T. C. (1991). Molecular characterization and immunogenicity of neutralization-sensitive Babesia bigemina merozoite surface proteins. Molecular and Biochemical Parasitology 47, 213222.
Mosqueda, J., McElwain, T. F., Stiller, D. and Palmer, G. H. (2002). Babesia bovis merozoite surface antigen 1 and rhoptry-associated protein 1 are expressed in sporozoites, and specific antibodies inhibit sporozoite attachment to erythrocytes. Infection and Immunity 70, 15991603.
Muhlnickel, C. J., Jefferies, R., Ryan, U. M. and Irwin, P. J. (2002). Babesia gibsoni infection in three dogs in Victoria. Australian Veterinary Journal 8, 606610.
Nichols, B. A., Chiappino, M. L. and O'Connor, G. R. (1983). Secretion from the rhoptries of Toxoplasma gondii during host-cell invasion. Journal of Ultrastructure Research 83, 8598.
Norimine, J., Mosqueda, J., Suarez, C., Palmer, G. H., McElwain, T. F., Mbassa, G. and Brown, W. C. (2003). Stimulation of T-helper cell gamma interferon and immunoglobulin G responses specific for Babesia bovis rhoptry-associated protein 1 (RAP-1) or a RAP-1 protein lacking the carboxy-terminal repeat region is insufficient to provide protective immunity against virulent B. bovis challenge. Infection and Immunity 71, 50215032.
Perkins, M. E. (1992). Rhoptry organelles of apicomplexan parasites. Parasitology Today 8, 2832.
Preiser, P., Kaviratne, M., Khan, S., Bannister, L. and Jarra, W. (2000). The apical organelles of malaria merozoites: host cell selection, invasion, host immunity and immune evasion. Microbes and Infection 12, 14611477.
Ridley, R. G., Takacs, B., Etlinger, H. and Scaife, J. G. (1990). A rhoptry antigen of Plasmodium falciparum is protective in Saimiri monkeys. Parasitology 101, 187192.
Sam-Yellowe, T. Y. (1996). Rhoptry organelles of the apicomplexa: their role in host cell invasion and intracellular survival. Parasitology Today 12, 308316.
Sam-Yellowe, T. Y., Shio, H., Perkins, M. E. (1988). Secretion of Plasmodium falciparum rhoptry protein into the plasma membrane of host erythrocytes. Journal of Cell Biology 106, 15071513.
Schofield, L., Bushell, G. R., Cooper, J. A., Saul, A. J., Upcroft, J. A. and Kidson, C. (1986). A rhoptry antigen of Plasmodium falciparum contains conserved and variable epitopes recognized by inhibitory monoclonal antibodies. Molecular and Biochemical Parasitology 18, 183195.
Skuce, P. J., Mallon, T. R. and Taylor, S. M. (1996). Molecular cloning of a putative rhoptry associated protein homologue from Babesia divergens. Molecular and Biochemical Parasitology 77, 99–102.
Suarez, C. E., McElwain, T. F., Echaide, I., Torioni de Echaide, S. and Palmer, G. H. (1994). Interstrain conservation of babesial RAP-1 surface-exposed B-cell epitopes despite rap-1 genomic polymorphism. Infection and Immunity 62, 35763579.
Suarez, C. E., McElwain, T. F., Stephens, E. B., Mishra, V. S. and Palmer, G. H. (1991 b). Sequence conservation among merozoite apical complex proteins of Babesia bovis, Babesia bigemina, and other apicomplexa. Molecular and Biochemical Parasitology 49, 329332.
Suarez, C. E., Palmer, G. H., Florin-Christensen, M., Hines, S. A., Hötzel, I. and McElwain, T. F. (2003). Organization, transcription, and expression of rhoptry associated protein genes in the Babesia bigemina rap-1 locus. Molecular and Biochemical Parasitology 127, 101112.
Suarez, C. E., Palmer, G. H., Jasmer, D. P., Hines, S. A., Perryman, L. E. and McElwain, T. F. (1991 a). Characterization of the gene encoding a 60-kilodalton Babesia bovis merozoite protein with conserved and surface exposed epitopes. Molecular and Biochemical Parasitology 46, 4552.
Sunaga, F., Namikawa, K. and Kanno, Y. (2002). Continuous in vitro culture of erythrocytic stages of Babesia gibsoni and virulence of the cultivated parasite. The Journal of Veterinary Medical Science 64, 571575.
Terkawi, M. A., Jia, H., Zhou, J., Lee, E. G., Igarashi, I., Fujisaki, K., Nishikawa, Y. and Xuan, X. (2007). Babesia gibsoni ribosomal phosphoprotein P0 induces cross-protective immunity against B. microti infection in mice. Vaccine 25, 20272035.
Wickramarachchi, T., Yengkhom, S. D., Asif, M. and Virander, S. C. (2008). Identification and characterization of a novel Plasmodium falciparum merozoite apical protein involved in erythrocyte binding and invasion. PLOS ONE 3, e1732.
Wright, I. G. (1991). Towards a synthetic Babesia vaccine. International Journal for Parasitology 21, 156159.
Wright, I. G., Casu, R., Commins, M. A., Dalrymple, B. P., Gale, K. R., Goodger, B. V., Riddles, P. W., Waltisbukl, D. J., Abetz, I., Berrie, D. A., Bowles, Y., Dimmock, C., Hayes, T., Kalnins, H., Leatch, G., McCrae, R., Montague, P. E., Nisbet, I. T., Parrodi, F., Peters, J. M., Scheiwe, P. C., Smith, W., Rode-Bramanis, K. and White, M. A. (1992). The development of a recombinant Babesia vaccine. Veterinary Parasitology 44, 313.
Yokoyama, N., Okamura, M. and Igarashi, I. (2006). Erythrocyte invasion by Babesia parasites: current advances in the elucidation of the molecular interactions between the protozoan ligands and host receptors in the invasion stage. Veterinary Parasitology 31, 2232.
Yokoyama, N., Suthisak, B., Hirata, H., Matsuo, T., Inoue, N., Sugimoto, C. and Igarashi, I. (2002). Cellular localization of Babesia bovis merozoite rhoptry-associated protein 1 and its erythrocyte-binding activity. Infection and Immunity 70, 58225826.
Zhou, J., Huang, B., Suzuki, H., Fujisaki, K., Igarashi, I. and Xuan, X. (2006 a). Isolation and identification of an actin gene from Babesia gibsoni. The Journal of Parasitology 92, 208210.
Zhou, J., Fukumoto, S., Jia, H., Yokoyama, N., Zhang, G., Fujisaki, K., Lin, J. and Xuan, X. (2006 b). Characterization of the Babesia gibsoni P18 as a homologue of thrombospondin related adhesive protein. Molecular and Biochemical Parasitology 148, 190198.
Zhou, J., Jia, H., Nishikawa, Y., Fujisaki, K. and Xuan, X. (2007). Babesia gibsoni rhoptry-associated protein 1 and its potential use as a diagnostic antigen. Veterinary Parasitology 145, 1620.

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