Importance of N-glycosylation and of GPI-anchored proteins in the invasion of bovine erythrocytes by Babesia bovis

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

Palermo (Italia)

Otro; Second Babesia World Summit; 2007

Instituto Zooprofilattico Sperimentale della Sicilia

Glycosylation, a post-translational addition of carbohydrate to proteins is ubiquitous in eukaryotic cells. In the majority of eukaryotic glycoproteins, the glycosyl residues are attached to the protein cores by N- and/or O- glycosidic bonds. In some proteins and glycoproteins, GPI anchors represent a distinctive type of carbohydrate modification.  The carbohydrate moieties of glycoproteins impart a wide range of properties to proteins and perform a number of roles in cellular functions. Glycosidic groups present in surface antigens of B.bovis have a relevant functional role. Some important vaccine candidates of the tick-transmitted bovine hemoparasite Babesia bovis are bound to the cell surface by glycosyl-phosphatidylinositol (GPI) anchors (Hines, S.A. et al., 1989, Molecular and Biochemical Parasitology.37:1-10). This is the case of the members of the Variable Merozoite Surface Antigen family (VMSA), which are homogenously distributed on the membrane of merozoites and sporozoites and contain neutralization-sensitive B-cell epitopes. These antigens have been postulated that are glycoproteins and to participate in 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. 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 culture medium 199/ bovine serum (6:4),  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. Parasite viability was assessed using fluorescence vital stains. To determine the importance of N-glycosylation in erythrocyte invasion, suspensions of B. bovis merozoites were treated with different concentrations of tunicamycin (inhibits N-glycosylation). After this treatment, merozoites were incubated with bovine erythrocytes in culture medium, and the parasitemia was determined in stained smears, after different time points. Our results showed 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. and can be observed a gradual reduction in parasitemia at day 3, with concentrations higher than 1uM of tunicamycin. Our results showed after 14 h a significant reduction in the numbers of infected erythrocytes 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 and the N- glycosylation for invasion of B. bovis merozoites, and provide relevant information for the design of subunit vaccines.