Nuclear Glycolytic Enzyme Enolase of Toxoplasma Gondii Functions as a Transcriptional Regulator

Apicomplexan parasites including Toxoplasma gondii have complex life cycles within different hosts and their infectivity relies on their capacity to regulate gene expression. However, little is known about the nuclear factors that regulate gene expression in these pathogens. Here, we report that T. gondii enolase TgENO2 is targeted to the nucleus of actively replicating parasites, where it specifically binds to nuclear chromatin in vivo. Using a ChIP-Seq technique, we provide evidence for TgENO2 enrichment at the 5′ untranslated gene regions containing the putative promoters of 241 nuclear genes. Ectopic expression of HA-tagged TgENO1 or TgENO2 led to changes in transcript levels of numerous gene targets. Targeted disruption of TgENO1 gene results in a decrease in brain cyst burden of chronically infected mice and in changes in transcript levels of several nuclear genes. Complementation of this knockout mutant with ectopic TgENO1-HA fully restored normal transcript levels. Our findings reveal that enolase functions extend beyond glycolytic activity and include a direct role in coordinating gene regulation in T. gondii

Characterization of a nuclear pore protein sheds light on the roles and composition of the Toxoplasma gondii nuclear pore complex

International audienceThe nuclear pore is a key structure in eukaryotes regulating nuclear-cytoplasmic transport as well as a wide range of cellular processes. Here, we report the characterization of the first Toxoplasma gondii nuclear pore protein, named TgNup302, which appears to be the orthologue of the mammalian Nup98-96 protein. We produced a conditional knock-down mutant that expresses TgNup302 under the control of an inducible tetracycline-regulated promoter. Under ATc treatment, a substantial decrease of TgNup302 protein in inducible knock-down (iKD) parasites was observed, causing a delay in parasite proliferation. Moreover, the nuclear protein TgENO2 was trapped in the cytoplasm of ATc-treated mutants, suggesting that TgNup302 is involved in nuclear transport. Fluorescence in situ hybridization revealed that TgNup302 is essential for 18S RNA export from the nucleus to the cytoplasm, while global mRNA export remains unchanged. Using an affinity tag purification combined with mass spectrometry, we identified additional components of the nuclear pore complex, including proteins potentially interacting with chromatin. Furthermore, reverse immunoprecipitation confirmed their interaction with TgNup302, and structured illuminated microscopy confirmed the NPC localization of some of the TgNup302-interacting proteins. Intriguingly, facilitates chromatin transcription complex (FACT) components were identified, suggesting the existence of an NPC-chromatin interaction in T. gondii. Identification of TgNup302-interacting proteins also provides the first glimpse at the NPC structure in Apicomplexa, suggesting a structural conservation of the NPC components between distant eukaryotes

Developmental expression of 4-repeat-Tau induces neuronal aneuploidy in Drosophila tauopathy models

Tau-mediated neurodegeneration in Alzheimer's disease and tauopathies is generally assumed to start in a normally developed brain. However, several lines of evidence suggest that impaired Tau isoform expression during development could affect mitosis and ploidy in post-mitotic differentiated tissue. Interestingly, the relative expression levels of Tau isoforms containing either 3 (3R-Tau) or 4 repeats (4R-Tau) play an important role both during brain development and neurodegeneration. Here, we used genetic and cellular tools to study the link between 3R and 4R-Tau isoform expression, mitotic progression in neuronal progenitors and post-mitotic neuronal survival. Our results illustrated that the severity of Tau-induced adult phenotypes depends on 4R-Tau isoform expression during development. As recently described, we observed a mitotic delay in 4R-Tau expressing cells of larval eye discs and brains. Live imaging revealed that the spindle undergoes a cycle of collapse and recovery before proceeding to anaphase. Furthermore, we found a high level of aneuploidy in post-mitotic differentiated tissue. Finally, we showed that overexpression of wild type and mutant 4R-Tau isoform in neuroblastoma SH-SY5Y cell lines is sufficient to induce monopolar spindles. Taken together, our results suggested that neurodegeneration could be in part linked to neuronal aneuploidy caused by 4R-Tau expression during brain development

A Novel Toxoplasma gondii Nuclear Factor TgNF3 Is a Dynamic Chromatin-Associated Component, Modulator of Nucleolar Architecture and Parasite Virulence

International audienceIn Toxoplasma gondii, cis-acting elements present in promoter sequences of genes that are stage-specifically regulated have been described. However, the nuclear factors that bind to these cis-acting elements and regulate promoter activities have not been identified. In the present study, we performed affinity purification, followed by proteomic analysis, to identify nuclear factors that bind to a stage-specific promoter in T. gondii. This led to the identification of several nuclear factors in T. gondii including a novel factor, designated herein as TgNF3. The N-terminal domain of TgNF3 shares similarities with the N-terminus of yeast nuclear FK506-binding protein (FKBP), known as a histone chaperone regulating gene silencing. Using anti-TgNF3 antibodies, HA-FLAG and YFP-tagged TgNF3, we show that TgNF3 is predominantly a parasite nucleolar, chromatin-associated protein that binds specifically to T. gondii gene promoters in vivo. Genome-wide analysis using chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) identified promoter occupancies by TgNF3. In addition, TgNF3 has a direct role in transcriptional control of genes involved in parasite metabolism, transcription and translation. The ectopic expression of TgNF3 in the tachyzoites revealed dynamic changes in the size of the nucleolus, leading to a severe attenuation of virulence in vivo. We demonstrate that TgNF3 physically interacts with H3, H4 and H2A/H2B assembled into bona fide core and nucleosome-associated histones. Furthermore, TgNF3 interacts specifically to histones in the context of stage-specific gene silencing of a promoter that lacks active epigenetic acetylated histone marks. In contrast to virulent tachyzoites, which express the majority of TgNF3 in the nucleolus, the protein is exclusively located in the cytoplasm of the avirulent bradyzoites. We propose a model where TgNF3 acts essentially to coordinate nucleolus and nuclear functions by modulating nucleosome activities during the intracellular proliferation of the virulent tachyzoites of T. gondii

Rab11A regulates dense granule transport and secretion during Toxoplasma gondii invasion of host cells and parasite replication

Toxoplasma gondii possesses an armada of secreted virulent factors that enable parasite invasion and survival into host cells. These factors are contained in specific secretory organelles, the rhoptries, micronemes and dense granules that release their content upon host cell recognition. Dense granules are secreted in a constitutive manner during parasite replication and play a crucial role in modulating host metabolic and immune responses. While the molecular mechanisms triggering rhoptry and microneme release upon host cell adhesion have been well studied, constitutive secretion remains a poorly explored aspect of T. gondii vesicular trafficking. Here, we investigated the role of the small GTPase Rab11A, a known regulator of exocytosis in eukaryotic cells. Our data revealed an essential role of Rab11A in promoting the cytoskeleton driven transport of dense granules and the release of their content into the vacuolar space. Rab11A also regulates transmembrane protein trafficking and localization during parasite replication, indicating a broader role of Rab11A in cargo exocytosis at the plasma membrane. Moreover, we found that Rab11A also regulates extracellular parasite motility and adhesion to host cells. In line with these findings, MIC2 secretion was altered in Rab11A-defective parasites, which also exhibited severe morphological defects. Strikingly, by live imaging we observed a polarized accumulation of Rab11A-positive vesicles and dense granules at the apical pole of extracellular motile and invading parasites suggesting that apically polarized Rab11A-dependent delivery of cargo regulates early secretory events during parasite entry into host cells

Utilización de herramientas de simulación en la robótica industrial

Análisis de herramienta de simulación utilizada en el entorno industrial y estudio de las ventajas de la programación "offline" en un entorno productivo. Programación de una célula de trabajo industrial y ampliación posterior de la misma utilizando la herramienta de simulación industrial

Ploidy of Cell-Sorted Trophic and Cystic Forms of Pneumocystis carinii

Once regarded as an AIDS-defining illness, Pneumocystis pneumonia (PcP) is nowadays prevailing in immunocompromised HIV-negative individuals such as patients receiving immunosuppressive therapies or affected by primary immunodeficiency. Moreover, Pneumocystis clinical spectrum is broadening to non-severely-immunocompromised subjects who could be colonized by the fungus while remaining asymptomatic for PcP, thus being able to transmit the infection by airborne route to susceptible hosts. Although the taxonomical position of the Pneumocystis genus has been clarified, several aspects of its life cycle remain elusive such as its mode of proliferation within the alveolus or its ploidy level. As no long-term culture model exists to grow Pneumocystis organisms in vitro, an option was to use a model of immunosuppressed rat infected with Pneumocystis carinii and sort life cycle stage fractions using a high-through-put cytometer. Subsequently, ploidy levels of the P. carinii trophic and cystic form fractions were measured by flow cytometry. In the cystic form, eight contents of DNA were measured thus strengthening the fact that each mature cyst contains eight haploid spores. Following release, each spore evolves into a trophic form. The majority of the trophic form fraction was haploid in our study. Some less abundant trophic forms displayed two contents of DNA indicating that they could undergo (i) mating/fusion leading to a diploid status or (ii) asexual mitotic division or (iii) both. Even less abundant trophic forms with four contents of DNA were suggestive of mitotic divisions occurring following mating in diploid trophic forms. Of interest, was the presence of trophic forms with three contents of DNA, an unusual finding that could be related to asymmetrical mitotic divisions occurring in other fungal species to create genetic diversity at lower energetic expenses than mating. Overall, ploidy data of P. carinii life cycle stages shed new light on the complexity of its modes of proliferation

Involvement of the Cytokine MIF in the Snail Host Immune Response to the Parasite Schistosoma mansoni

We have identified and characterized a Macrophage Migration Inhibitory Factor (MIF) family member in the Lophotrochozoan invertebrate, Biomphalaria glabrata, the snail intermediate host of the human blood fluke Schistosoma mansoni. In mammals, MIF is a widely expressed pleiotropic cytokine with potent pro-inflammatory properties that controls cell functions such as gene expression, proliferation or apoptosis. Here we show that the MIF protein from B. glabrata (BgMIF) is expressed in circulating immune defense cells (hemocytes) of the snail as well as in the B. glabrata embryonic (Bge) cell line that has hemocyte-like features. Recombinant BgMIF (rBgMIF) induced cell proliferation and inhibited NO-dependent p53-mediated apoptosis in Bge cells. Moreover, knock-down of BgMIF expression in Bge cells interfered with the in vitro encapsulation of S. mansoni sporocysts. Furthermore, the in vivo knock-down of BgMIF prevented the changes in circulating hemocyte populations that occur in response to an infection by S. mansoni miracidia and led to a significant increase in the parasite burden of the snails. These results provide the first functional evidence that a MIF ortholog is involved in an invertebrate immune response towards a parasitic infection and highlight the importance of cytokines in invertebrate-parasite interactions

Imagerie Multimodale par Cartographie 3D en excitation pulsée : de la cellule au tissu

To repair, supply or regenerate deficient organs, the bioengineering field consists of synthesising functionalised biomaterials. To visualise the synthesised structures in a non invasive way and in physiological conditions, new imaging techniques tend to be developed. Among them, microscopy associating multiphoton excitation with fluorescence detection or Second Harmonic Generation enables a visualisation in depth of extracellular matrix structures, without any exogenous dye. The first part of this work was to characterise multiphoton absorption probability in function of different excitation conditions, that means a modulation of the excitation beam through two different systems (Cavity Dumper and EOM). In a second part, we implemented and optimised the detection of the SHG signal coming from collagen in biological tissues. Through SHG measurements, we showed modifications occurring on the collagen network of the extracellular matrix of cartilage, when sample were submitted to mechanical (compression) or biochemical (enzymatic) constraint. We also were interested by the vascular research field and showed the ability of multiphoton microscopy to image without any fluorescent dye, the elastin network of the media and the collagen network of the adventice. We could appreciate qualitatively the effect of cryopreservation or fixation on the arterial wall, and the remodelling of a substitute implemented in a rabbit to supply its carotid. A last biological application concerned study of tumors, and showed us the complementarities between a macroscopic study with information obtained by microscopy.L'un des enjeux de la bioingénierie consiste à synthétiser des biomatériaux visant à régénérer, remplacer ou suppléer des organes déficients. Afin de visualiser des structures dans des conditions proches de la réalité physiologique, et de manière non invasive, de nouvelles techniques d'imagerie ne cessent de se développer. En particulier, la microscopie associant une excitation multiphoton et la détection de signaux de fluorescence et de signaux de SHG (Second Harmonic Generation) permet l'observation en profondeur de composants de matrices extracellulaires sans marqueur fluorescent exogène. Ce travail basé sur ces techniques a permis dans un premier temps d'apprécier les probabilités d'absorption multiphoton en fonction de la modulation de l'excitation avec deux systèmes différents (Cavity Dumper et EOM). Une seconde partie de ce projet a été consacrée à la mise en place et l'optimisation de la détection de signaux SHG provenant du collagène dans les tissus biologiques. Nous avons utilisé ces méthodes d'imagerie pour mettre en évidence les modifications intervenant au niveau des réseaux de collagène de la MEC de cartilage suite à l'application d'une contrainte mécanique (compression) ou biochimique (enzymatique). Puis, nous nous sommes intéressés au domaine vasculaire, en montrant la possibilité d'imager sans marquage fluorescent les réseaux d'élastine de la média (en mettant à profit son autofluorescence) et le réseau de collagène de l'adventice (générant un fort signal SHG). Nous avons ainsi pu apprécier l'état des structures en fonction de différentes conditions de préservation (congélation, fixation) et le remodelage de substituts artériels implantés chez le lapin. Enfin, une dernière application biologique, basée sur l'étude de tumeurs, nous montre la complémentarité et l'intérêt d'une imagerie de type macroscopique avec les diverses modalités de détection en microscopie

3D Multimodal imaging with pulsed excitation : From cell to tissue

L’un des enjeux de la bioingénierie consiste à synthétiser des biomatériaux visant à régénérer, remplacer ou suppléer des organes déficients. Afin de visualiser des structures dans des conditions proches de la réalité physiologique, et de manière non invasive, de nouvelles techniques d’imagerie ne cessent de se développer. En particulier, la microscopie associant une excitation multiphoton et la détection de signaux de fluorescence et de signaux de SHG (Second Harmonic Generation) permet l’observation en profondeur de composants de matrices extracellulaires sans marqueur fluorescent exogène. Ce travail basé sur ces techniques a permis dans un premier temps d’apprécier les probabilités d’absorption multiphoton en fonction de la modulation de l’excitation avec deux systèmes différents (Cavity Dumper et EOM). Une seconde partie de ce projet a été consacrée à la mise en place et l’optimisation de la détection de signaux SHG provenant du collagène dans les tissus biologiques. Nous avons utilisé ces méthodes d’imagerie pour mettre en évidence les modifications intervenant au niveau des réseaux de collagène de la MEC de cartilage suite à l’application d’une contrainte mécanique (compression) ou biochimique (enzymatique). Puis, nous nous sommes intéressés au domaine vasculaire, en montrant la possibilité d’imager sans marquage fluorescent les réseaux d’élastine de la média (en mettant à profit son autofluorescence) et le réseau de collagène de l’adventice (générant un fort signal SHG). Nous avons ainsi pu apprécier l’état des structures en fonction de différentes conditions de préservation (congélation, fixation) et le remodelage de substituts artériels implantés chez le lapin. Enfin, une dernière application biologique, basée sur l’étude de tumeurs, nous montre la complémentarité et l’intérêt d’une imagerie de type macroscopique avec les diverses modalités de détection en microscopie.To repair, supply or regenerate deficient organs, the bioengineering field consists of synthesising functionalised biomaterials. To visualise the synthesised structures in a non invasive way and in physiological conditions, new imaging techniques tend to be developed. Among them, microscopy associating multiphoton excitation with fluorescence detection or Second Harmonic Generation enables a visualisation in depth of extracellular matrix structures, without any exogenous dye. The first part of this work was to characterise multiphoton absorption probability in function of different excitation conditions, that means a modulation of the excitation beam through two different systems (Cavity Dumper and EOM). In a second part, we implemented and optimised the detection of the SHG signal coming from collagen in biological tissues. Through SHG measurements, we showed modifications occurring on the collagen network of the extracellular matrix of cartilage, when sample were submitted to mechanical (compression) or biochemical (enzymatic) constraint. We also were interested by the vascular research field and showed the ability of multiphoton microscopy to image without any fluorescent dye, the elastin network of the media and the collagen network of the adventice. We could appreciate qualitatively the effect of cryopreservation or fixation on the arterial wall, and the remodelling of a substitute implemented in a rabbit to supply its carotid. A last biological application concerned study of tumors, and showed us the complementarities between a macroscopic study with information obtained by microscopy