Relevance of GPI-Anchored Antigens for the Survival of Babesia bovis Merozoites

A.E. RODRÍGUEZ; I. ECHAIDE; E. BARAVALLE; L. SCHNITTGER; M. FLORIN-CHRISTENSEN

Mérida (México)

Conferencia; 9th Biennial Conference of Society for Tropical Veterinary Medicine; 2007

Society for Tropical Veterinary Medicine

Some important vaccine candidates of the tick-transmitted bovine hemoparasite Babesia bovis are bound to the cell surface by glycosyl-phosphatidylinositol (GPI) anchors. This is the case of the members of the Variable Merozoite Surface Antigen family, which are homogenously distributed on the membrane of merozoites and sporozoites and contain neutralization-sensitive B-cell epitopes. These antigens have been postulated to participate in the the processes of recognition and/or attachment to the erythrocyte membrane. In other apicomplexan protozoa, such as Plasmodium falciparum, GPI anchored proteins play a critical role in the survival of the parasites. In this work, we have tested if this is also the case for B. bovis analyzing the overall importance of GPI-anchored antigens by two approaches. First, B. bovis merozoites were cultivated in vitro in the presence of different concentrations of mannosamine, an inhibitor of the formation of GPI anchors, and parasitemia was evaluated in Giemsa stained smears after 72 h. Our results show a significant (p < 0.01) decrease in the proportions of infected erythrocytes at manosammine concentrations higher than 1 mM, similar to what was observed by other authors for Plasmodium sp. Second, in vitro cultured B. bovis merozoites, purified by differential centrifugation, were incubated with phosphatidylinositol (PI)-specific phospholipase C (1 U/ml) for 1 h, at 37oC in 60% culture medium 199 and 40% bovine serum, after which merozoites were washed with the same mix and exposed to bovine erythrocytes. Aliquots were removed at different time points and parasitemia was counted in Giemsa-stained smears. Negative controls consisted on merozoite suspensions treated with buffer and no enzyme. PI-phospholipase C is known to cleave the phosphodiester bond in the GPI anchor, thus releasing attached proteins. Our results showed a significant reduction in the numbers of infected erythrocytes after 14 h in the phospholipase C-treated samples as compared to negative controls. Fluorescein diacetate vital staining showed that the observed effects were not due to a viability decrease in the enzyme-treated samples. These experiments are the first to demonstrate the importance of GPI-anchored surface antigens for invasion of B. bovis merozoites, and provide relevant information for the design of subunit vaccines.