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.].

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

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. IMPORTANCET. 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.

The autophagy machinery restrains iNKT cell activation through CD1D1 internalization.

Invariant natural killer T (iNKT) cells are innate T cells with powerful immune regulatory functions that recognize glycolipid antigens presented by the CD1D protein. While iNKT cell-activating glycolipids are currently being explored for their efficacy to improve immunotherapy against infectious diseases and cancer, little is known about the mechanisms that control CD1D antigen presentation and iNKT cell activation in vivo. CD1D molecules survey endocytic pathways to bind lipid antigens in MHC class II-containing compartments (MIICs) before recycling to the plasma membrane. Autophagosomes intersect with MIICs and autophagy-related proteins are known to support antigen loading for increased CD4+ T cell immunity. Here, we report that mice with dendritic cell (DC)-specific deletion of the essential autophagy gene Atg5 showed better CD1D1-restricted glycolipid presentation in vivo. These effects led to enhanced iNKT cell cytokine production upon antigen recognition and lower bacterial loads during Sphingomonas paucimobilis infection. Enhanced iNKT cell activation was independent of receptor-mediated glycolipid uptake or costimulatory signals. Instead, loss of Atg5 in DCs impaired clathrin-dependent internalization of CD1D1 molecules via the adaptor protein complex 2 (AP2) and, thus, increased surface expression of stimulatory CD1D1-glycolipid complexes. These findings indicate that the autophagic machinery assists in the recruitment of AP2 to CD1D1 molecules resulting in attenuated iNKT cell activation, in contrast to the supporting role of macroautophagy in CD4+ T cell stimulation.

3D Structure and Interaction of p24ß and p24δ Golgi Dynamics Domains: Implication for p24 Complex Formation and Cargo Transport.

The p24 family consists of four subfamilies (p24α, p24ß, p24γ, and p24δ), and the proteins are thought to form hetero-oligomeric complexes for efficient transport of cargo proteins from the endoplasmic reticulum to the Golgi apparatus. The proteins possess a conserved luminal Golgi dynamics (GOLD) domain, whose functions are largely unknown. Here, we present structural and biochemical studies of p24ß1 and p24δ1 GOLD domains. Use of GOLD domain-deleted mutants revealed that the GOLD domain of p24δ1 is required for proper p24 hetero-oligomeric complex formation and efficient transport of GPI-anchored proteins. The p24ß1 and p24δ1 GOLD domains share a common ß-sandwich fold with a characteristic intrasheet disulfide bond. The GOLD domain of p24δ1 crystallized as dimers, allowing the analysis of a homophilic interaction site. Surface plasmon resonance and solution NMR analyses revealed that p24ß1 and p24δ1 GOLD domains interact weakly (Kd= ~10-4M). Bi-protein titration provided interaction site maps. We propose that the heterophilic interaction of p24 GOLD domains contributes to the formation of the p24 hetero-oligomeric complex and to efficient cargo transport.

The α-helical region in p24γ2 subunit of p24 protein cargo receptor is pivotal for the recognition and transport of glycosylphosphatidylinositol-anchored proteins.

Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are group of proteins that depend on p24 cargo receptors for their transport from the endoplasmic reticulum to the Golgi apparatus. The GPI anchor is expected to act as a sorting and transport signal, but so far little is known about the recognition mechanism. In the present study we investigate the GPI-AP transport in cell knockdown of p24γ, the most diverse p24 subfamily. Knockdown of p24γ2 but not of other p24γ family members impaired the transport of a reporter GPI-AP. Restoration of the knockdown-induced phenotype using chimeric constructs between p24γ2 and the related p24γ1 further implied a role of the α-helical region of p24γ2 but not its GOLD domain in the specific binding of GPI-APs. We conclude that motifs in the membrane-adjacent α-helical region of p24γ2 are involved in recognition of GPI-APs and are consequently responsible for the incorporation of these proteins into coat protein complex II-coated transport vesicles.

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 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.