Identification and characterisation of aTheileria annulataproline-rich microtubule and SH3 domain-interacting protein (TaMISHIP) that forms a complex with CLASP1, EB1, and CD2AP at the schizont surface

Abstract

Theileria annulata is an apicomplexan parasite that modifies the phenotype of its host cell completely, inducing uncontrolled proliferation, resistance to apoptosis, and increased invasiveness. The infected cell thus resembles a cancer cell, and changes to various host cell signalling pathways accompany transformation. Most of the molecular mechanisms leading to Theileria‐induced immortalization of leukocytes remain unknown. The parasite dissolves the surrounding host cell membrane soon after invasion and starts interacting with host proteins, ensuring its propagation by stably associating with the host cell microtubule network. By using BioID technology together with fluorescence microscopy and co‐immunoprecipitation, we identified a CLASP1/CD2AP/EB1‐containing protein complex that surrounds the schizont throughout the host cell cycle and integrates bovine adaptor proteins (CIN85, 14‐3‐3 epsilon, and ASAP1). This complex also includes the schizont membrane protein Ta‐p104 together with a novel secreted T. annulata protein (encoded by TA20980), which we term microtubule and SH3 domain‐interacting protein (TaMISHIP). TaMISHIP localises to the schizont surface and contains a functional EB1‐binding SxIP motif, as well as functional SH3 domain‐binding Px(P/A)xPR motifs that mediate its interaction with CD2AP. Upon overexpression in non‐infected bovine macrophages, TaMISHIP causes binucleation, potentially indicative of a role in cytokinesis.

Dynamic methylation of histone H3K18 in differentiating Theileria parasites

“…S3]. We confirmed the presence of methylated and acetylated H3K18 in TBL3, TpMD409 and TaC12 27 Supplementary Fig. S2b].…”

Theileria secretes proteins to subvert its host leukocyte

“…Theileria schizonts are decorated by two host cell proteins, CD2 associated protein (CD2AP) [Huber et al., 2018] and CLIP‐170‐associating protein 1 (CLASP1) during all leukocyte cell cycle steps [Huber et al., 2017]. This is in contrast to host cell EB1 [Woods et al., 2013b] and (Polo‐like kinase) Plk1 [von Schubert et al., 2010], where binding to parasite surface varies.…”

Theileria’s Strategies and Effector Mechanisms for Host Cell Transformation: From Invasion to Immortalization

“…The striking localization of CLASP1 at the schizont surface meant that the kinetochore binding fragment of CLASP1 served as an effective bait for proximity ligation pull-down experiments to identify further host-parasite interactions. These experiments led to the identification of a number of host adaptor proteins that coat the schizont surface including CD2 associated protein (CD2AP), c-Cbl-interacting protein of 85 kDa (CIN85) and Arf-GAP with SHD domain ANK repeat and PH domain-containing protein 1 (ASAP1) (Huber et al, 2018). Adaptor proteins are proteins that contain several protein-binding domains that facilitate the formation of large signaling complexes (Flynn, 2001).…”

“…CD2AP and CIN85 are also recruited by Toxoplasma to the moving junction during invasion, and invasion-deficient T. gondii mutants with depleted rhoptry proteins RON2, RON4, and RON5 fail to recruit CD2AP/CIN85 (Guérin et al, 2017). However, it is unlikely that CD2AP and CIN85 play any role in Theileria invasion because these proteins were not detected at the parasite surface until development into a schizont had started (Huber et al, 2018).…”

“…Host CD2AP, CIN85 and ASAP1 were found to form a complex at the parasite surface together with host CLASP1 and EB1, T. annulata p104 and a novel T. annulata encoded protein named T. annulata proline rich microtubule and SH-domain interaction protein (TaMISHIP, TA20980, Huber et al, 2018). TaMISHIP is a protein that contains a signal peptide and predicted cleavage site, is highly disordered, and is unique to transforming species of Theileria.…”