Dirk A. E. Dobbelaere

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 cell's 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. The apicomplexan parasite Theileria can reprogram the cell it infects, inducing uncontrolled proliferation and clonal expansion. This is brought about by the schizont, which resides free in the host cell cytoplasm. As the schizont never leaves the cell to infect other cells, it can only persist provided it is distributed over the two daughter cells each time the host cell divides. This is achieved by interacting dynamically with microtubules (MTs) that form part of the host cell mitotic apparatus. How MTs are recruited to the schizont surface is not known. MTs are highly dynamic, undergoing continuous cycles of growth and shrinkage that is regulated to a large extent by an array of proteins, called +TIPs, that associate with the free plus-ends of MTs. End-binding protein 1 (EB1) is a master regulator and central adaptor that mediates MT plus-end tracking of potentially all other +TIPs. We established that a schizont surface protein, p104, provides a docking site for EB1, which critically depends on a consensus SxIP motif, present in p104. These finding provides important new insight into the complex interaction of the transforming schizont with host cell MTs. To our knowledge, p104 is the first pathogen-derived protein identified so far to join the SxIP family of EB1-binding proteins.

Theileria parvais a tick-transmitted protozoan parasite that infects and transforms bovine lymphocytes. We have previously shown thatTheileria parvaChitongo is an isolate with a lower virulence than that ofT. parvaMuguga. Lower virulence appeared to be correlated with a delayed onset of the logarithmic growth phase ofT. parvaChitongo-transformed peripheral blood mononuclear cells afterin vitroinfection. In the current study, infection experiments with WC1⁺γδ T cells revealed that onlyT. parvaMuguga could infect these cells and that no transformed cells could be obtained withT. parvaChitongo sporozoites. Subsequent analysis of the susceptibility of different cell lines and purified populations of lymphocytes to infection and transformation by both isolates showed thatT. parvaMuguga sporozoites could attach to and infect CD4⁺, CD8⁺, and WC1⁺T lymphocytes, butT. parvaChitongo sporozoites were observed to bind only to the CD8⁺T cell population. Flow cytometry analysis of established, transformed clones confirmed this bias in target cells.T. parvaMuguga-transformed clones consisted of different cell surface phenotypes, suggesting that they were derived from either host CD4⁺, CD8⁺, or WC1⁺T cells. In contrast, allin vitroandin vivoT. parvaChitongo-transformed clones expressed CD8 but not CD4 or WC1, suggesting that theT. parvaChitongo-transformed target cells were exclusively infected CD8⁺lymphocytes. Thus, a role of cell tropism in virulence is likely. Since the adhesion molecule p67 is 100% identical between the two strains, a second, high-affinity adhesin that determines target cell specificity appears to exist.

Despite a significant growth in food production over the past half-century, one of the most important challenges facing society today is how to feed an expected population of some nine billion by the middle of the 20th century. To meet the expected demand for food without significant increases in prices, it has been estimated that we need to produce 70–100 per cent more food, in light of the growing impacts of climate change, concerns over energy security, regional dietary shifts and the Millennium Development target of halving world poverty and hunger by 2015. The goal for the agricultural sector is no longer simply to maximize productivity, but to optimize across a far more complex landscape of production, rural development, environmental, social justice and food consumption outcomes. However, there remain significant challenges to developing national and international policies that support the wide emergence of more sustainable forms of land use and efficient agricultural production. The lack of inform...

The protozoan parasite Theileria inhabits the host cell cytoplasm and possesses the unique capacity to transform the cells it infects, inducing continuous proliferation and protection against apoptosis. The transforming schizont is a multinucleated syncytium that resides free in the host cell cytoplasm and is strictly intracellular. To maintain transformation, it is crucial that this syncytium is divided over the two daughter cells at each host cell cytokinesis. This process was dissected using different cell cycle synchronization methods in combination with the targeted application of specific inhibitors. We found that Theileria schizonts associate with newly formed host cell microtubules that emanate from the spindle poles, positioning the parasite at the equatorial region of the mitotic cell where host cell chromosomes assemble during metaphase. During anaphase, the schizont interacts closely with host cell central spindle. As part of this process, the schizont recruits a host cell mitotic kinase, Polo-like kinase 1, and we established that parasite association with host cell central spindles requires Polo-like kinase 1 catalytic activity. Blocking the interaction between the schizont and astral as well as central spindle microtubules prevented parasite segregation between the daughter cells during cytokinesis. Our findings provide a striking example of how an intracellular eukaryotic pathogen that evolved ways to induce the uncontrolled proliferation of the cells it infects usurps the host cell mitotic machinery, including Polo-like kinase 1, one of the pivotal mitotic kinases, to ensure its own persistence and survival. As part of their survival tactics, intracellular parasites often resort to cunning mechanisms to manipulate the cells they inhabit. Theileria, an important and particularly artful parasite of cattle in the tropics, transforms parasitized cells (that is, it induces continuous proliferation and protection from apoptosis—a state reminiscent of tumor cells). As a large, strictly intracellular syncytium, the transforming Theileria schizont cannot exit from the infected cell to invade other target cells. How then does the parasite ensure that each daughter cell, generated upon host cell division, remains infected and transformed? Our data show that the parasite co-opts the mitotic apparatus of the host cell and Plk1, a host protein kinase with a central regulatory role in mitosis and cytokinesis. As the host cell enters mitosis, the schizont binds to the microtubules that emanate symmetrically from the two spindle poles. This microtubule binding positions the schizont so that it spans the equatorial region of the mitotic cell where host cell chromosomes assemble. Then, as sister chromatids start to separate, the schizont associates with Plk1 and the central spindle that assembles between the separating chromosomes, with the activity of Plk1 presumably coordinating progression through mitosis with proper schizont positioning. This alignment with the central spindle positions the schizont to be included in the plane of cell division at the onset of cytokinesis, thus ensuring faithful passage of a Theileria schizont on to each daughter cell.

Apicomplexan parasites within the genus Theileria have the ability to induce continuous proliferation and prevent apoptosis of the infected bovine leukocyte. Protection against apoptosis involves constitutive activation of the bovine transcription factor NF‐κB in a parasite‐dependent manner. Activation of NF‐κB is thought to involve recruitment of IKK signalosomes at the surface of the macroschizont stage of the parasite, and it has been postulated that additional host proteins with adaptor or scaffolding function may be involved in signalosome formation. In this study two clonal cell lines were identified that show marked differences in the level of activated NF‐κB. Further characterization of these lines demonstrated that elevated levels of activated NF‐κB correlated with increased resistance to cell death and detection of parasite‐associated IKK signalosomes, supporting results of our previous studies. Evidence was also provided for the existence of host‐ and parasite‐dependent NF‐κB activation pathways that are influenced by the architecture of the actin cytoskeleton. Despite this influence, it appears that the primary event required for formation of the parasite‐dependent IKK signalosome is likely to be an interaction between a signalosome component and a parasite‐encoded surface ligand.

The intracellular protozoan parasite Theileria parva transforms bovine lymphocytes inducing uncontrolled proliferation. Proteins released from the parasite are assumed to contribute to phenotypic changes of the host cell and parasite persistence. With 85 members, genes encoding subtelomeric variable secreted proteins (SVSPs) form the largest gene family in T. parva. The majority of SVSPs contain predicted signal peptides, suggesting secretion into the host cell cytoplasm. We analysed SVSP expression in T. parva-transformed cell lines established in vitro by infection of T or B lymphocytes with cloned T. parva parasites. Microarray and quantitative real-time PCR analysis revealed mRNA expression for a wide range of SVSP genes. The pattern of mRNA expression was largely defined by the parasite genotype and not by host background or cell type, and found to be relatively stable in vitro over a period of two months. Interestingly, immunofluorescence analysis carried out on cell lines established from a cloned parasite showed that expression of a single SVSP encoded by TP03_0882 is limited to only a small percentage of parasites. Epitope-tagged TP03_0882 expressed in mammalian cells was found to translocate into the nucleus, a process that could be attributed to two different nuclear localisation signals. Our analysis reveals a complex pattern of Theileria SVSP mRNA expression, which depends on the parasite genotype. Whereas in cell lines established from a cloned parasite transcripts can be found corresponding to a wide range of SVSP genes, only a minority of parasites appear to express a particular SVSP protein. The fact that a number of SVSPs contain functional nuclear localisation signals suggests that proteins released from the parasite could contribute to phenotypic changes of the host cell. This initial characterisation will facilitate future studies on the regulation of SVSP gene expression and the potential biological role of these enigmatic proteins.