The function of the p53 protein as the central effector molecule of the p53 apoptotic pathway was investigated in a reversible model of epigenetic transformation. The infection of bovine leukocytes by the intracellular protozoan parasite Theileria annulata results in parasitedependent transformation and proliferation of the host cells. We found p53 to be largely localized in the host cell cytoplasm and associated with the parasite membrane of isolated schizonts. Curing infected cells of the parasite with the theilericidal drug buparvaquone resulted in a time-dependent translocation of p53 into the host cell nucleus and the upregulation of the proapoptotic Bax and Apaf-1 and the downregulation of the anti-apoptotic Bcl-2 proteins. Although buparvaquone treatment led to apoptosis of the host cell, inhibition of either p53 or Bax significantly reduced buparvaquone-induced apoptosis of the transformed cells. Thus, the p53 apoptotic pathway of host cells is not induced by infection and transformation with Theileria by a mechanism involving cytoplasmic sequestration of p53. The close association of host cell p53 with the parasite membrane implies that the parasite either interacts directly with p53 or mediates cytoplasmic sequestration of p53 by interacting with other host cell proteins regulating p53 localization.

Leucoproliferative Theileria parasites possess the unique capability to transform their bovine host cell, resulting in tumour‐like characteristics like uncontrolled proliferation. The molecular mechanisms underlying this parasite‐dependent process are only poorly understood. In the current study, bioinformatic analysis of the Theileria annulata surface protein (TaSP) from different T. annulata isolates identified a conserved CDK1 phosphorylation motif T131PTK within the extracellular, polymorphic domain of TaSP. Phosphorylation assays with radioactively labelled ATP as well as ELISA‐based experiments using a phospho‐threonine‐proline (pThr‐Pro) antibody revealed, that CDK1‐cyclin B specifically phosphorylates T131, identifying TaSP as a substrate in vitro. Confocal microscopy and proximity ligation assays suggest an interaction between CDK1 and TaSP in T. annulata‐infected cells. Further studies demonstrated a nearly complete co‐localization of the pThr‐Pro signal and TaSP only in cells in interphase, pointing towards a cell cycle‐dependent event. Immunostainings of isolated, non‐permeabilized schizonts confirmed the presence of the pThr‐Pro epitope on the schizont's surface. Lambda phosphatase treatment abolished the pThr‐Pro signal of the schizont, which was reconstituted by the addition of CDK1‐cyclin B. Treatment of T. annulata‐infected cells with the CDK1 inhibitor purvalanol A resulted in morphological changes characterized by tubulin‐rich cell protrusions and an extension of the schizont, and a dose‐dependent reduction of BrdU incorporation and Ki67 staining of T. annulata‐infected cells, demonstrating a clear impact on the Theileria‐dependent proliferation of the bovine host cell. Our data reveal the parasite surface protein TaSP as a target for the host cell kinase CDK1, a major player during cell division. Targeting the uncontrolled proliferation of Theileria‐infected cells is a novel and reasonable approach to limit parasite load in order to facilitate a successful cellular immune response against the parasite.

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