Theileria annulatasurface protein (TaSP) is a target of cyclin‐dependent kinase 1 phosphorylation in Theileria annulata‐infected cells

Abstract

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.

Screening and identification of Theileria annulata subtelomere-encoded variable secreted protein-950454 (SVSP454) interacting proteins from bovine B cells

“…Therefore, investigating the interactions of T. annulata and its host provides insights into T. annulata -induced pathogenesis. Although some molecules have been reported to affect T. annulata -induced transformation, metabolism, and invasion by regulating host signaling-hubs [ 8 – 10 , 21 ], the evidence for the relevant roles of secreted multifamily (SVSP)-host interactions has not been presented until now. Herein, SVSP454, a member of the SVSP multigene family, was used as bait to explore its interacting molecules in host cells.…”

Theileria annulata SVSP455 interacts with host HSP60

“…In Theileria-induced transformation, several host cell signaling pathways are activated that are involved in controlling parasite proliferation and survival, including the NF-kB, c-Jun N-terminal kinase (JNK) and c-Myc signaling pathways [1,[17][18][19], but the exact mechanism remains unclear. Decades of research has resulted in the identification of many parasite proteins that contribute to transformation, including TaPIN1 [20,21], TaSP [22,23] and Ta-p104 [24]. However, the molecules that play a key role in transformation remain unclear.…”

Theileria secretes proteins to subvert its host leukocyte

“…This motif (T 131 PTK) is exposed to host cell cytosol and is only phosphorylated in interphase cells. Further experiments showed that TaSP interacts with CDK1 and drug inhibition of CDK1 altered the morphology of T. annulate ‐infected cells and caused a reduction in their proliferation, adding more evidence to the role of TaSP in transformation [Mackiewicz et al., 2020].…”