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Infection and Immunity, October 2002, p. 5822-5826, Vol. 70, No. 10
0019-9567/02/$04.00+0     DOI: 10.1128/IAI.70.10.5822-5826.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Cellular Localization of Babesia bovis Merozoite Rhoptry-Associated Protein 1 and Its Erythrocyte-Binding Activity

Naoaki Yokoyama, Boonchit Suthisak, Haruyuki Hirata, Tomohide Matsuo, Noboru Inoue, Chihiro Sugimoto, and Ikuo Igarashi*

National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido 080-8555, Japan

Received 30 April 2002/ Returned for modification 7 June 2002/ Accepted 28 June 2002

The cellular localization of Babesia bovis rhoptry-associated protein 1 (RAP-1) and its erythrocyte-binding affinity were examined with anti-RAP-1 antibodies. In an indirect immunofluorescent antibody test, RAP-1 was detectable in all developmental stages of merozoites and in extracellular merozoites. In the early stage of merozoite development, RAP-1 appears as a dense accumulation, which later thins out and blankets the host cell cytoplasm, but retains a denser mass around newly formed parasite nuclei. The preferential accumulations of RAP-1 on the inner surface of a host cell membrane and bordering the parasite's outer surface were demonstrable by immunoelectron microscopy. An erythrocyte-binding assay with the lysate of merozoites demonstrated RAP-1 binding to both bovine and equine erythrocytes. Anti-RAP-1 monoclonal antibody 1C1 prevented the interaction of RAP-1 with bovine erythrocytes and significantly inhibited parasite proliferation in vitro. With the recombinant RAP-1, the addition of increasing concentrations of Ca2+ accentuated its binding affinity with bovine erythrocytes. The present findings lend support to an earlier proposition of an erythrocytic binding role for RAP-1 expressed in B. bovis merozoites and, possibly, its involvement in the escape of newly formed merozoites from host cells.

* Corresponding author. Mailing address: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan. Phone: 81-155-49-5641. Fax: 81-155-49-5643. E-mail: igarcpmi{at}obihiro.ac.jp.

Editor: W. A. Petri, Jr.

Infection and Immunity, October 2002, p. 5822-5826, Vol. 70, No. 10
0019-9567/02/$04.00+0     DOI: 10.1128/IAI.70.10.5822-5826.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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