Abstract
In this thesis we investigated the invasion of erythrocytes taking place during the asexual erythrocytic blood stage of the apicomplexan parasites Babesia bovis parasite.
Host cell invasion by apicomplexan parasites is a complex process requiring multiple receptor-ligand interactions, involving association of the merozoite with the erythrocyte surface, reorientation and attachment
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of the apical end of the merozoite. In parasites like Toxoplasma gondii and Plasmodium falciparum many proteins have been identified that are involved in invasion including proteins present on the merozoite surface and proteins present in secretory organelles located at the apical end of the merozoite. An in vitro invasion assay of B. bovis was established to be able to characterize the mechanisms and the proteins involved in invasion. Invasion of liberated merozoites is completed in 1 h in phosphate buffered saline solution. The manipulation of intracellular calcium concentration or actin polymerization in the merozoite inhibits invasion indicating that B. bovis invades erythrocytes in a similar way as Plasmodium merozoites. Proteins that are secreted into the environment during in vitro invasion can be detected directly by protein staining after 2D gel electrophoresis. This in vitro invasion assay was used to assess the host-specificity of B. bovis. In vivo, B. bovis infections have only been observed in bovine species. In vitro, human, ovine, porcine, equine and caprine erythrocytes were all invaded by B. bovis. Human erythrocytes were invaded more efficiently than bovine erythrocytes, whereas goat erythrocytes were invaded at very low level. Significant differences in invasion efficiency into erythrocytes from different individuals of the same species were observed. Besides invasion, intracellular duplication of B. bovis parasites also took place in all erythrocyte species, except for goats. Only in bovine erythrocytes subsequent rounds of invasion were observed. B. bovis merozoite invasion was shown to be dependent on sialic-acid residues present on the host cell. Pre-incubation of merozoites with n-acetylneuraminyl-lactose decreased invasion efficiency by ~45%, whereas addition prior to invasion had no significant effect. Thus invasion might be dependent on the presence of merozoite membrane proteins already accessible during pre-incubation prior to invasion.
Only two proteins (AMA-1 and TRAP) involved in invasion of other apicomplexans were identified from a large B. bovis EST database and these were sequenced and characterized. B. bovis invasion was inhibited by antiserum raised against peptides from several domains of AMA-1. Antisera raised against domain III of AMA-1 give the strongest invasion inhibition. B. bovis TRAP was surprisingly found in merozoites. In Plasmodium species TRAP has been found in the sporozoite stage. Antibodies directed against BbTRAP inhibited invasion. Immunofluorescence showed that B. bovis AMA-1 and TRAP are localized at the apical region of the merozoites. P. falciparum AMA-1 and TRAP proteins are known to be cleaved off and secreted into environment during invasion. 1D and 2D-gel electrophoresis of total merozoites extract and invasion supernatant followed by immunoblotting with B. bovis AMA-1 antisera showed that this protein is cleaved from 82 kDa to a 69 kDa and secreted into the surrounding environment. B. bovis TRAP also was found in invasion supernatant.
Transfection of in malaria parasites provides a valuable tool for analyzing gene function. An attempt was done to establish a transfection system of B. bovis with dhfr as a selectable marker. The B. bovis dhfr-ts genomic locus and cDNA was cloned and sequenced. Beside the dhfr-ts gene of 2.83 kb, four other genes were identified in a 7.41 kb region of which at least three are expressed during the erythrocytic cycle. Three of the genes were highly conserved in closely related Theileria species and gene synteny was observed between B. bovis and T. parva. B. bovis merozoite cultures electroporated with plasmids containing T. gondii dhfr-tsm2m3 or human dhfr under control of B. bovis dhfr regions did not produce stable transfected parasites. Transient expression using GFP, YFP or ß-galactosidase as a reporter gene was also not yet established. Nevertheless, parasite cultures with decreased sensitivity for pyrimethamine and WR99210 were repeatly isolated. The dhfr gene of five resistant cultures was sequenced and showed to contain a S125F amino acid substitution. Homology modelling of B. bovis DHFR by using the P. falciparum DHFR structure as a template, suggested that S125F affects the binding site of NADPH. The five resistant cultures that were first selected on either pyrimethamine or WR99210 turned out to be cross-resistant for both drugs. Comparable drug sensitivities were only observed in a quadruple mutant of P. falciparum dhfr displaying strong resistance to pyrimethamine and 10-fold enhanced resistance against WR99210. Wildtype B. bovis was similar to this mutant at 3 of the 4 mutated positions possibly explaining its low sensitivity for pyrimethamine.
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