Sandra Huber

Recruitment of EB1, a Master Regulator of Microtubule Dynamics, to the Surface of the Theileria annulata Schizont

The apicomplexan parasite Theileria annulata transforms infected host cells, inducing uncontrolled proliferation and clonal expansion of the parasitized cell population. Shortly after sporozoite entry into the target cell, the surrounding host cell membrane is dissolved and an array of host cell microtubules (MTs) surrounds the parasite, which develops into the transforming schizont. The latter does not egress to invade and transform other cells. Instead, it remains tethered to host cell MTs and, during mitosis and cytokinesis, engages the cells astral and central spindle MTs to secure its distribution between the two daughter cells. The molecular mechanism by which the schizont recruits and stabilizes host cell MTs is not known. MT minus ends are mostly anchored in the MT organizing center, while the plus ends explore the cellular space, switching constantly between phases of growth and shrinkage (called dynamic instability). Assuming the plus ends of growing MTs provide the first point of contact with the parasite, we focused on the complex protein machinery associated with these structures. We now report how the schizont recruits end-binding protein 1 (EB1), a central component of the MT plus end protein interaction network and key regulator of host cell MT dynamics. Using a range of in vitro experiments, we demonstrate that T. annulata p104, a polymorphic antigen expressed on the schizont surface, functions as a genuine EB1-binding protein and can recruit EB1 in the absence of any other parasite proteins. Binding strictly depends on a consensus SxIP motif located in a highly disordered C-terminal region of p104. We further show that parasite interaction with host cell EB1 is cell cycle regulated. This is the first description of a pathogen-encoded protein to interact with EB1 via a bona-fide SxIP motif. Our findings provide important new insight into the mode of interaction between Theileria and the host cell cytoskeleton.

A 1‐Year Controlled Clinical Trial of Immediate Implants Placed in Fresh Extraction Sockets: Stability Measurements and Crestal Bone Level Changes

PURPOSE The aim of this study was to measure stability and crestal bone level changes of implants placed in fresh extraction sockets in elderly patients. METHODS Thirty-five patients who were in need of tooth extractions were recruited for this study. They received a total of 65 implants in both jaws to support fixed or removable prostheses. The teeth were carefully extracted, the implants set directly in the root socket, and resonance frequency analysis (RFA) measurements were simultaneously performed (Time 1=T1). After a healing time of 6 to 10weeks the measurements were repeated (Time 2=T2). Orthograd periapical radiographs were taken when the new prostheses were fabricated and after 1year of loaded period. The distance between the first visible bone implant contact (BIC) and the implant-shoulder was measured and crestal bone loss was calculated (ΔBIC). Mean RFA and BIC were compared for various subgroups (p<.05). By means of a fixed effects model, the impact of the parameters gender, jaw, and prosthetic indication on RFA measurements was analyzed (p<.016). RESULTS The mean implant stability quotient (ISQ) values were 64.4±6.7 at T1 and 64.0±at 8.6T2, with a trend to higher values for male patients. The mixed model showed that only the jaw had a statistically significant impact on ISQ values, with higher values for the mandible. Mean crestal bone loss was small with 0.49±0.81mm, ranging form 0.1 to 2.4mm. Twenty percent of the implant sites lost more than 1-mm crestal bone. No differences were found in subgroups. CONCLUSIONS Good primary and secondary stability of the implants was reached in both jaws. Crestal bone loss was small but may not be fully predictable for a single site. This treatment modality can be applied successfully in elderly patients and can be suggested for various prosthetic indications in both jaws.

The Microtubule-Stabilizing Protein CLASP1 Associates with the Theileria annulata Schizont Surface via Its Kinetochore-Binding Domain.

T. annulata, the only eukaryote known to be capable of transforming another eukaryote, is a widespread parasite of veterinary importance that puts 250 million cattle at risk worldwide and limits livestock development for some of the poorest people in the world. Crucial to the pathology of Theileria is its ability to interact with host microtubules and the mitotic spindle of the infected cell. This study builds on our previous work in investigating the host and parasite molecules involved in mediating this interaction. Because it is not possible to genetically manipulate Theileria schizonts, identifying protein interaction partners is critical to understanding the function of parasite proteins. By identifying two Theileria surface proteins that are involved in the interaction between CLASP1 and the parasite, we provide important insights into the molecular basis of Theileria persistence within a dividing cell. ABSTRACT Theileria is an apicomplexan parasite whose presence within the cytoplasm of a leukocyte induces cellular transformation and causes uncontrolled proliferation and clonal expansion of the infected cell. The intracellular schizont utilizes the host cell’s own mitotic machinery to ensure its distribution to both daughter cells by associating closely with microtubules (MTs) and incorporating itself within the central spindle. We show that CLASP1, an MT-stabilizing protein that plays important roles in regulating kinetochore-MT attachment and central spindle positioning, is sequestered at the Theileria annulata schizont surface. We used live-cell imaging and immunofluorescence in combination with MT depolymerization assays to demonstrate that CLASP1 binds to the schizont surface in an MT-independent manner throughout the cell cycle and that the recruitment of the related CLASP2 protein to the schizont is MT dependent. By transfecting Theileria-infected cells with a panel of truncation mutants, we found that the kinetochore-binding domain of CLASP1 is necessary and sufficient for parasite localization, revealing that CLASP1 interaction with the parasite occurs independently of EB1. We overexpressed the MT-binding domain of CLASP1 in parasitized cells. This exhibited a dominant negative effect on host MT stability and led to altered parasite size and morphology, emphasizing the importance of proper MT dynamics for Theileria partitioning during host cell division. Using coimmunoprecipitation, we demonstrate that CLASP1 interacts, directly or indirectly, with the schizont membrane protein p104, and we describe for the first time TA03615, a Theileria protein which localizes to the parasite surface, where it has the potential to participate in parasite-host interactions. IMPORTANCE T. annulata, the only eukaryote known to be capable of transforming another eukaryote, is a widespread parasite of veterinary importance that puts 250 million cattle at risk worldwide and limits livestock development for some of the poorest people in the world. Crucial to the pathology of Theileria is its ability to interact with host microtubules and the mitotic spindle of the infected cell. This study builds on our previous work in investigating the host and parasite molecules involved in mediating this interaction. Because it is not possible to genetically manipulate Theileria schizonts, identifying protein interaction partners is critical to understanding the function of parasite proteins. By identifying two Theileria surface proteins that are involved in the interaction between CLASP1 and the parasite, we provide important insights into the molecular basis of Theileria persistence within a dividing cell.

Identification and characterization of a Theileria annulata proline rich microtubule and SH3-domain interacting protein (TaMISHIP) that forms a complex with CLASP1, EB1 and CD2AP at the schizont surface

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.

Erratum for Huber et al., “The Microtubule-Stabilizing Protein CLASP1 Associates with the Theileria annulata Schizont Surface via Its Kinetochore-Binding Domain”

[This corrects the article DOI: 10.1128/mSphere.00215-17.].