What do politicians think of the common consolidated corporate tax base? A Belgian case study

This paper analyzes the views of Belgian politicians on the Common Consolidated Corporate Tax Base (CCCTB), an ambitious project to harmonize corporate taxation in the EU. Applying case study methodology, the results show that most politicians were proponents of this new tax system. During the discussions in several political institutions, the politicians referred to the macro-economic impact, the legal certainty and their party’s view to found their opinion. Besides several agreements, certain aspects of CCCTB like the optionality and the applied tax rate involved clear differences in view between left and right-wing politicians, which could hamper a political agreement on CCCTB

A glycosylphosphatidylinositol-anchored carbonic anhydrase-related protein of Toxoplasma gondii is important for rhoptry biogenesis and virulence

Carbonic anhydrase-related proteins (CARPs) have previously been described as catalytically inactive proteins closely related to α-carbonic anhydrases (α-CAs). These CARPs are found in animals (both vertebrates and invertebrates) and viruses as either independent proteins or domains of other proteins. We report here the identification of a new CARP (TgCA_RP) in the unicellular organism Toxoplasma gondii that is related to the recently described η-class CA found in Plasmodium falciparum. TgCA_RP is posttranslationally modified at its C terminus with a glycosylphosphatidylinositol anchor that is important for its localization in intracellular tachyzoites. The protein localizes throughout the rhoptry bulbs of mature tachyzoites and to the outer membrane of nascent rhoptries in dividing tachyzoites, as demonstrated by immunofluorescence and immunoelectron microscopy using specific antibodies. T. gondii mutant tachyzoites lacking TgCA_RP display a growth and invasion phenotype in vitro and have atypical rhoptry morphology. The mutants also exhibit reduced virulence in a mouse model. Our results show that TgCA_RP plays an important role in the biogenesis of rhoptries

Plasmodium falciparum enolase: stage-specific expression and sub-cellular localization

<p>Abstract</p> <p>Background</p> <p>In an earlier study, it was observed that the vaccination with <it>Plasmodium falciparum </it>enolase can confer partial protection against malaria in mice. Evidence has also build up to indicate that enolases may perform several non-glycolytic functions in pathogens. Investigating the stage-specific expression and sub-cellular localization of a protein may provide insights into its moonlighting functions.</p> <p>Methods</p> <p>Sub-cellular localization of <it>P. falciparum </it>enolase was examined using immunofluorescence assay, immuno-gold electron microscopy and western blotting.</p> <p>Results</p> <p>Enolase protein was detected at every stage in parasite life cycle examined. In asexual stages, enolase was predominantly (≥85–90%) present in soluble fraction, while in sexual stages it was mostly associated with particulate fraction. Apart from cytosol, enolase was found to be associated with nucleus, food vacuole, cytoskeleton and plasma membrane.</p> <p>Conclusion</p> <p>Diverse localization of enolase suggests that apart from catalyzing the conversion of 2-phosphoglycericacid into phosphoenolpyruvate in glycolysis, enolase may be involved in a host of other biological functions. For instance, enolase localized on the merozoite surface may be involved in red blood cell invasion; vacuolar enolase may be involved in food vacuole formation and/or development; nuclear enolase may play a role in transcription.</p

In Vivo Biotinylation of the Toxoplasma Parasitophorous Vacuole Reveals Novel Dense Granule Proteins Important for Parasite Growth and Pathogenesis.

UnlabelledToxoplasma gondii is an obligate intracellular parasite that invades host cells and replicates within a unique parasitophorous vacuole. To maintain this intracellular niche, the parasite secretes an array of dense granule proteins (GRAs) into the nascent parasitophorous vacuole. These GRAs are believed to play key roles in vacuolar remodeling, nutrient uptake, and immune evasion while the parasite is replicating within the host cell. Despite the central role of GRAs in the Toxoplasma life cycle, only a subset of these proteins have been identified, and many of their roles have not been fully elucidated. In this report, we utilize the promiscuous biotin ligase BirA* to biotinylate GRA proteins secreted into the vacuole and then identify those proteins by affinity purification and mass spectrometry. Using GRA-BirA* fusion proteins as bait, we have identified a large number of known and candidate GRAs and verified localization of 13 novel GRA proteins by endogenous gene tagging. We proceeded to functionally characterize three related GRAs from this group (GRA38, GRA39, and GRA40) by gene knockout. While Δgra38 and Δgra40 parasites showed no altered phenotype, disruption of GRA39 results in slow-growing parasites that contain striking lipid deposits in the parasitophorous vacuole, suggesting a role in lipid regulation that is important for parasite growth. In addition, parasites lacking GRA39 showed dramatically reduced virulence and a lower tissue cyst burden in vivo Together, the findings from this work reveal a partial vacuolar proteome of T. gondii and identify a novel GRA that plays a key role in parasite replication and pathogenesis.ImportanceMost intracellular pathogens reside inside a membrane-bound vacuole within their host cell that is extensively modified by the pathogen to optimize intracellular growth and avoid host defenses. In Toxoplasma, this vacuole is modified by a host of secretory GRA proteins, many of which remain unidentified. Here we demonstrate that in vivo biotinylation of proximal and interacting proteins using the promiscuous biotin ligase BirA* is a powerful approach to rapidly identify vacuolar GRA proteins. We further demonstrate that one factor identified by this approach, GRA39, plays an important role in the ability of the parasite to replicate within its host cell and cause disease

A novel dense granule protein, GRA41, regulates timing of egress and calcium sensitivity in Toxoplasma gondii

Toxoplasma gondii is an obligate intracellular apicomplexan parasite with high seroprevalence in humans. Repeated lytic cycles of invasion, replication, and egress drive both the propagation and the virulence of this parasite. Key steps in this cycle, including invasion and egress, depend on tightly regulated calcium fluxes and, although many of the calcium-dependent effectors have been identified, the factors that detect and regulate the calcium fluxes are mostly unknown. To address this knowledge gap, we used a forward genetic approach to isolate mutants resistant to extracellular exposure to the calcium ionophore A23187. Through whole genome sequencing and complementation, we have determined that a nonsense mutation in a previously uncharacterised protein is responsible for the ionophore resistance of one of the mutants. The complete loss of this protein recapitulates the resistance phenotype and importantly shows defects in calcium regulation and in the timing of egress. The affected protein, GRA41, localises to the dense granules and is secreted into the parasitophorous vacuole where it associates with the tubulovesicular network. Our findings support a connection between the tubulovesicular network and ion homeostasis within the parasite, and thus a novel role for the vacuole of this important pathogen

Systematic characterization of all Toxoplasma gondii TBC domain-containing proteins identifies an essential regulator of Rab2 in the secretory pathway.

Toxoplasma gondii resides in its intracellular niche by employing a series of specialized secretory organelles that play roles in invasion, host cell manipulation, and parasite replication. Rab GTPases are major regulators of the parasite's secretory traffic that function as nucleotide-dependent molecular switches to control vesicle trafficking. While many of the Rab proteins have been characterized in T. gondii, precisely how these Rabs are regulated remains poorly understood. To better understand the parasite's secretory traffic, we investigated the entire family of Tre2-Bub2-Cdc16 (TBC) domain-containing proteins, which are known to be involved in vesicle fusion and secretory protein trafficking. We first determined the localization of all 18 TBC domain-containing proteins to discrete regions of the secretory pathway or other vesicles in the parasite. Second, we use an auxin-inducible degron approach to demonstrate that the protozoan-specific TgTBC9 protein, which localizes to the endoplasmic reticulum (ER), is essential for parasite survival. Knockdown of TgTBC9 results in parasite growth arrest and affects the organization of the ER and mitochondrial morphology. TgTBC9 knockdown also results in the formation of large lipid droplets (LDs) and multi-membranous structures surrounded by ER membranes, further indicating a disruption of ER functions. We show that the conserved dual-finger active site in the TBC domain of the protein is critical for its GTPase-activating protein (GAP) function and that the Plasmodium falciparum orthologue of TgTBC9 can rescue the lethal knockdown. We additionally show by immunoprecipitation and yeast 2 hybrid analyses that TgTBC9 preferentially binds Rab2, indicating that the TBC9-Rab2 pair controls ER morphology and vesicular trafficking in the parasite. Together, these studies identify the first essential TBC protein described in any protozoan and provide new insight into intracellular vesicle trafficking in T. gondii

Vacuolar and plasma membrane stripping and autophagic elimination of Toxoplasma gondii in primed effector macrophages

Apicomplexan protozoan pathogens avoid destruction and establish a replicative niche within host cells by forming a nonfusogenic parasitophorous vacuole (PV). Here we present evidence for lysosome-mediated degradation of Toxoplasma gondii after invasion of macrophages activated in vivo. Pathogen elimination was dependent on the interferon γ inducible-p47 GTPase, IGTP, required PI3K activity, and was preceded by PV membrane indentation, vesiculation, disruption, and, surprisingly, stripping of the parasite plasma membrane. Denuded parasites were enveloped in autophagosome-like vacuoles, which ultimately fused with lysosomes. These observations outline a series of mechanisms used by effector cells to redirect the fate of a classically nonfusogenic intracellular pathogen toward a path of immune elimination