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.
Apical membrane antigen 1 (AMA-1) is a micronemal protein secreted to the surface of merozoites of Plasmodium species and Toxoplasma gondii tachyzoites in order to fulfill an essential but noncharacterized function in host cell invasion. Here we describe cloning and characterization of a Babesia bovis AMA-1 homologue designated BbAMA-1. The overall level of similarity of BbAMA-1 to P. falciparum AMA-1 was low (18%), but characteristic features like a transmembrane domain near the C terminus, a predicted short cytoplasmic C-terminal sequence with conserved sequence properties, and an extracellular domain containing 14 conserved cysteine residues putatively involved in disulfide bridge formation are typical of AMA-1. Rabbit polyclonal antisera were raised against three synthetic peptides derived from the N-terminal region and domains II and III of the putative extracellular domain and were shown to recognize specifically recombinant BbAMA-1 expressed in Escherichia coli. Immunofluorescence microscopy showed that there was labeling of the apical half of merozoites with these antisera. Preincubation of free merozoites with all three antisera reduced the efficiency of invasion of erythrocytes by a maximum of 65%. Antisera raised against the N-terminal peptide detected a 82-kDa protein on Western blots and a 69-kDa protein in the supernatant that was harvested after in vitro invasion, suggesting that proteolytic processing and secretion take place during or shortly after invasion. A combination of two-dimensional Western blotting and metabolic labeling allowing direct identification of spots reacting with the BbAMA-1 peptide antisera together with the very low silver staining intensity of these spots indicated that very low levels of BbAMA-1 are present in Babesia merozoites.
Gaffar
Fasila R
FR
Department of Infectious Diseases and Immunology, Utrecht University, 3508 TD, Utrecht, The Netherlands.
Yatsuda
Ana P
AP
Franssen
Frits F J
FF
de Vries
Erik
E
D000595:Amino Acid Sequence; D000818:Animals; D000913:Antibodies, Protozoan; D000953:Antigens, Protozoan / Q000235:genetics / Q000276:immunology*; D016793:Babesia bovis / Q000235:genetics / Q000276:immunology* / Q000472:pathogenicity*; D001404:Babesiosis / Q000097:blood / Q000276:immunology / Q000469:parasitology; D001483:Base Sequence; D002417:Cattle; D002418:Cattle Diseases / Q000097:blood / Q000276:immunology / Q000469:parasitology; D018076:DNA, Complementary / Q000235:genetics; D016054:DNA, Protozoan / Q000235:genetics; D004912:Erythrocytes / Q000469:parasitology; D066298:In Vitro Techniques; D008565:Membrane Proteins / Q000276:immunology*; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D015800:Protozoan Proteins / Q000235:genetics / Q000276:immunology*; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity
D000913:Antibodies, Protozoan; D000953:Antigens, Protozoan; D018076:DNA, Complementary; D016054:DNA, Protozoan; D008565:Membrane Proteins; D015800:Protozoan Proteins; D011994:Recombinant Proteins; C090077:apical membrane antigen I, Plasmodium