Glycosylphosphatidylinositol of Babesia bovis: studies on its structure,

RODRIGUEZ A.E.; COUTO, A S; ECHAIDE, I.; SCHNITTGER, L; FLORIN-CHRISTENSEN, M

Rosario-Argentina

Congreso; VIII Congreso Argentino de Protozoología y Enfermedades Parasitarias; 2008

Sociedad Argentina de Protozoología

Glycerophosphatidylinositol (GPI) molecules have been found to play important roles in the biology and pathogenicity of several protozoon parasites. They serve as membrane anchors of surface antigens and are also present as free precursors in the cell membrane. In Babesia bovis, GPI anchors have been previously demonstrated in merozoite surface antigens that were identified as vaccine candidates. Since most aspects of GPI biochemistry are unknown for this parasite, we have started the structural analysis of its free GPI molecules. The glycolipid fraction, containing free GPIs, was extracted from a suspension of in vitro cultured merozoites and separated by thin layer chromatography. The different spots were eluted and treated with phosphatidylinositol-specific phospholipase C (PI-PLC) followed by two-phase lipid extraction to separate GPI glycan and lipid constituents. GPI glycans were radioactively labeled, separated by ion exchange chromatography and detected by scintillation counting in the eluate fractions. As a control, an erythrocyte ghost suspension containing comparable amounts of protein was analyzed in parallel. Our results revealed the occurrence of a B. bovis-specific free GPI species, containing an additional mannose as compared to those observed in bovine erythrocytes. Analysis of the lipid moiety of this GPI showed a glycerophospholipid structure, containing at least two different fatty acid species: one of C20 and another of an undetermined size between C14 and C16, possibly corresponding to a modified fatty acid. To further understand the GPI biosynthetic capabilities of B. bovis, we searched the recently sequenced genome of this parasite and found seven genes that are homologous to those encoding for well characterized GPI biosynthetic enzymes of P. falciparum: PIG-A, DPM1, PIG-O, GAA-1, PIG-M, GPI-1 and GPI-8. These genes are not clustered and are distributed among the four B. bovis chromosomes. Finally, the biological importance of GPI biosynthesis was studied using mannosamine, an inhibitor of this process. B. bovis merozoites were impaired to grow in vitro in the presence of concentrations of 1 mM and higher, similarly to what has been shown with P. falciparum. Our work might lead to an increased understanding of the pathogenicity of bovine babesiosis and to the development of improved control strategies. (Supported by INCO 003691-MEDLABAB; and PIP 5580, CONICET, Argentina. AER received a Doctoral Fellowship from CONICET and AC and MFC are CONICET Researchers)