Initiation of translation and cellular localization of Theileria annulata casein kinase IIalpha: implication for its role in host cell transformation.

Theileria annulata and T. parva are protozoa that infect bovine leukocytes which leads to subsequent transformation and uncontrolled proliferation of these cells. It has been proposed that the CKIIalpha subunit of T. parva induces mitogenic pathways of host leukocytes by being exported into the host cell. The evidence for this is the existence of a predicted N-terminal secretion signal-like peptide. We tested this hypothesis by analyzing gene structure, translation, and protein localization of the T. annulata CKIIalpha (TaCKIIalpha). The determined TaCKIIalpha-ORF potentially codes for a 50 kDa protein with an N-terminal extension including a possible signal sequence not present in CKIIalpha proteins of non-Theileria species. However, antisera raised against TaCKIIalpha recognized a protein of a molecular weight of about 40 kDa and, therefore, inconsistent with this predicted molecular weight. We demonstrate by in vitro transcription/translation that this discrepancy is due to translation from a downstream initiation site omitting the putative N-terminal signal sequence and thus excluding the notion that the protein product is secreted via the classical secretory pathway. In corroboration immunofluorescence investigations suggest that the TaCKIIalpha subunit is confined to the parasite schizonts within the host cell. On the basis of the above findings it seems highly unlikely that export via the classical pathway of the parasite CKIIalpha is the way in which this protein possibly contributes to host cell transformation.

Characterization of a polymorphic Theileria annulata surface protein (TaSP) closely related to PIM of Theileria parva: implications for use in diagnostic tests and subunit vaccines.

Theileria annulata is a tick-transmitted protozoan that causes tropical theileriosis, an often fatal leukoproliferative disorder of cattle. To characterize and identify parasite proteins suitable as diagnostic antigens and/or vaccine candidates, a cDNA clone encoding a macroschizont stage protein was isolated and characterized (here designated TaSP). The gene, present as a single copy within the parasite genome, is transcribed in the sporozoite and schizont stage and codes for a protein of about 315 amino acids, having a predicted molecular weight of 36 kDa. Allelic variants were found within single parasite isolates and between isolates originating from different geographical regions. The N-terminal part contains a predicted signal peptide and the C-terminal section encodes membrane-spanning regions. Comparison of a number of cDNA clones showed that both these sequence regions are conserved while the central region shows both size and amino acid sequence polymorphism. High identity of the N- and C-terminal regions with the polymorphic immunodominant molecule (PIM) of Theileria parva (identity of 93%), the existence of a central polymorphic region and two short introns within genomic clones suggest that the presented gene/protein may be the T. annulata homologue of PIM. However, the central region of TaSP has no significant identity with PIM, contains no repetitive peptide motifs and is shorter, resulting in a lower molecular weight. The existence of the predicted secretion signal peptide and membrane spanning regions suggest that TaSP is located at the parasite membrane.