Analysis of Cell Cycle Associated Proteins from Theileria annulata

The protozoan parasite, Theileria annulata, is a tick transmitted intracellular pathogen of domestic cattle in tropical regions of Africa and Asia. The parasite life cycle within the bovine host involves proliferative phases within the host lymphocytes and erythrocytes. As a means of characterising cdc2-related kinases (CRKs) involved in these stages which would control the parasite division cycle, p13sucl affinity columns have been used to show binding and activity of parasite proteins which are likely to have a role in the cell cycle. Further experiments showed the existence of at least two p13sucl binding kinases in T. annulata, together with possible substrate specificities of these polypeptides. Another gene involved in replication and division has been isolated, namely the large subunit of ribonucleotide reductase (Rl) which encodes a unique N terminal extension to the predicted amino acid sequence compared to higher eukaryotes. The role of this extension is unknown but could serve in the allosteric regulation of the parasite enzyme. It is also feasible that this region could be used as a target for peptidomimetic inhibitors specific for the parasite enzyme. The Rl polypeptide immunolocalises to the parasite nuclei whereas the host Rl remains in the cytoplasm. Using anti-sera generated against a range of host and parasite cell cycle associated proteins it was possible to gain a picture of changes occuring during the intracellular differentiation of the parasite within the lymphocyte. These results show that the intracellular differentiation initially involves both host and parasite cell cycles but there is an early decline in the amounts of two polypeptide markers of the host cell cycle whilst the parasite continues to undergo repeated nuclear division prior to the differentiation into the merozoite stage. Attempts were made to synchronise infected cells using aphidicolin and the effects of this drug on the expression of cell cycle associated genes in T. annulata was also examined