Molecular characterizations of three distinct Babesia gibsoni rhoptry-associated protein-1s (RAP-1s).
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.
Terkawi
M A
MA
National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan.
Amornthep
A
A
Ooka
H
H
Aboge
G
G
Jia
H
H
Goo
Y-K
YK
Nelson
B
B
Yamagishi
J
J
Nishikawa
Y
Y
Igarashi
I
I
Kawazu
S-I
SI
Fujisaki
K
K
Xuan
X
X
D000818:Animals; D001403:Babesia / Q000145:classification / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D001404:Babesiosis / Q000175:diagnosis / Q000469:parasitology / Q000662:veterinary*; D015153:Blotting, Western; D016054:DNA, Protozoan / Q000032:analysis; D004283:Dog Diseases / Q000175:diagnosis* / Q000469:parasitology; D004285:Dogs; D004797:Enzyme-Linked Immunosorbent Assay; D005455:Fluorescent Antibody Technique; D051379:Mice; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D015800:Protozoan Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D017422:Sequence Analysis, DNA
D016054:DNA, Protozoan; D015800:Protozoan Proteins; C097551:rhoptry-associated protein-1, Babesia