Amina Bittame - Academia.edu (original) (raw)

Papers by Amina Bittame

Conclusions et perspectives 8 Conclusion Annexe Solomon Sea circulation and water mass modificati... more Conclusions et perspectives 8 Conclusion Annexe Solomon Sea circulation and water mass modifications : response at ENSO timescales Bibliographie Listes des figures Listes des tables La mer des Salomon : enjeux scientifiques La mer des Salomon est située dans l'océan Pacifique tropical occidental, au nordest de l'Australie, entre la Papouasie Nouvelle-Guinée à l'ouest, les îles Salomon à l'est, et l'archipel Bismarck au nord. C'est une mer semi-fermée avec une topographie très complexe constituée de chaînes d'îles de tailles différentes. Elle communique avec l'océan équatorial via les détroits de Vitiaz, de Salomon et le chenal St George. La mer des Salomon constitue la zone de transit final des eaux de l'océan Pacifique tropical sud dans leur retour vers l'équateur. La variabilité de la circulation en mer des Salomon est d'une importance cruciale dans une perspective climatique car le système de courant dans cette zone fait partie du système des cellules subtropicales et ramène les eaux subductées du Elles concernent également tous les aspects de variabilité à méso et possiblement sousmésoéchelles qui sont observés dans cette mer et dont une partie de l'origine est lié à la présence d'un fort courant de bord-ouest. La mer des Salomon : enjeux méthodologiques Ce travail de thèse a avant tout pour objectif, la mise en oeuvre d'une configuration de modèle permettant la résolution de nombreuses échelles océaniques, notamment la Ainsi, les motivations méthodologiques sous-jacentes à cette thèse sont la mise en oeuvre d'un modèle à la résolution du 1/36 • afin de représenter de façon assez réaliste la dynamique associée à la bathymétrie complexe de la région, la dynamique des mésoéchelles propres à cette région et d'aborder une première résolution des sous-mésoéchelles. Enfin, dans la dernière partie nous présentons deux applications du modèle à haute résolution construit. Dans la perspective SWOT, notre modèle est utilisé pour effectuer une étude de faisabilité d'inversion d'image de traceur (chapitre 6) en vue de la correction de la dynamique. Nous avons aussi exploré les effets d'"upscaling" d'un environnement régional sur un environnement grande échelle i.e. les impacts de la présence du modèle 1/36 • (échelle Première partie Contexte scientifique 9 Chapitre 1

Cell Reports, Dec 1, 2015

Highlights d T. gondii GRA2 and GRA6 shape tubules of the intravacuolar network (IVN) d GRA6 asso... more Highlights d T. gondii GRA2 and GRA6 shape tubules of the intravacuolar network (IVN) d GRA6 association with PV membranes promotes MHC I antigen presentation d GRA6 binding to IVN limits its MHC I antigen presentation in vitro and in vivo d Membranes of T. gondii IVN may play a role in immune modulation

Bio-protocol, 2016

Dissecting the interactions established between proteins and membranes in a given type of cells i... more Dissecting the interactions established between proteins and membranes in a given type of cells is not an easy task. Using a cell-free system of large unilamellar vesicles (LUVs) to analyze these interactions may help decipher these interactions and identify potential membrane deformations induced by the proteins incubated with these LUVs. This article describes the protocols for 1) extraction of total lipids from eukaryotic cells using the method developed by Bligh and Dyer (1959), 2) the quantification of glycerophospholipids by gas chromatography after methanolysis, followed by 3) the formation of LUVs by extrusion, 4) protein-lipid binding assay, 5) analysis of the incubation product by transmission electron microscopy (TEM) and by flotation across a discontinuous sucrose gradient and finally, 6) analysis of the proteins by immunoblot and revelation of the glycerophospholipids by iodin fumigation. [Background] Cell-free systems consisting in giant unilamellar vesicles (GUVs; vesicles composed of a single bilayer of phospholipids and with a diameter greater than 1 μm) or liposomes incubated with recombinant proteins may help understand these interactions. Depending on their diameter and number of lamellae, liposomes are classified into small unilamellar vesicles (SUVs; vesicles constituted of a single bilayer of phospholipids and with a diameter comprised between 20 and 100 nm), large unilamellar vesicles (LUVs; vesicles constituted of a single bilayer of phospholipids and with a diameter comprised between 100 and 400 nm), large multilamellar vesicles (MLVs; vesicles constituted of multiple phospholipid bilayers and with a diameter comprised between 200 nm and 3 μm) and multivesicular vesicles (MVVs; large vesicles composed of a single bilayer of phospholipids and containing several smaller vesicles, each composed of a single bilayer of phospholipids).

HAL (Le Centre pour la Communication Scientifique Directe), May 30, 2010

BIO-PROTOCOL, 2016

Cell Reports, 2015

The parasitophorous vacuole in which Toxoplasma gondii develops is characterized by a membranous ... more The parasitophorous vacuole in which Toxoplasma gondii develops is characterized by a membranous nanotubular network (MNN) that connects the parasites together and to the parasitophorous vacuole membrane. The molecular components, the formation and the function of the MNN remain poorly explored. We had previously demonstrated indirectly, by the study of the phenotype of knocked-out parasites, that both the dense granule secreted proteins GRA2 and GRA6, which contain three amphipathic alpha-helices and one long hydrophobic alpha-helix, respectively, are key elements of the MNN formation (Mercier et al., 2002; Travier et al., 2008). The aim of this work was to demonstrate directly the role of both these proteins in the MNN formation, using an in vitro system to study the proteins-membranes interactions. Recombinant GRA2 (rGRA2) and GRA6 (rGRA6) proteins were purified from Escherichia coli. Dynamic light scattering and circular dichroism showed that rGRA2, which folds with an alpha-helical pattern, is purified as two soluble complexes, a potential dimer and complexes of higher molecular weight. rGRA6, which folds partially with an alpha-helical pattern, is purified as monomers. When incubated with Large Unilamellar Vesicles (LUVs) formed with complex lipids, rGRA2 associated preferentially with the membranes of 100 nm diameter-LUVs and induced the formation of short membranous tubules observed by transmission electron microscopy (TEM). rGRA6 also associated with 100 nm diameter-LUVs and tethered vesicles. Upon co-incubation of rGRA2 and rGRA6 with LUVs, the frequency of tubule formation did increase. These results thus validate the cooperation of both GRA2 and GRA6 to deform membranes and to generate membranous tubules.

Within the host cell, the parasitophorous vacuole in which Toxoplasma gondii develops, is charact... more Within the host cell, the parasitophorous vacuole in which Toxoplasma gondii develops, is characterized by a membranous nanotubular network (MNN). The molecular components, the formation and the function of the MNN remain poorly explored.We had previously demonstrated indirectly that the secreted dense granule GRA2 protein, which contains three amphipathic alpha helices, is a key element of the MNN formation (Mercier et al., 2002; Travier et al., 2008). The aim of this work was to demonstrate directly the role of GRA2 in the MNN formation, using an in vitro system to study the GRA2-membrane interactions. Native and recombinant GRA2 proteins were puri ed from Toxoplasma and from Escherichia coli, respectively. Dynamic light scattering and circular dichroism showed that recombinant GRA2, which folds with an alpha-helical pattern, is puri ed as both a potential dimer and complexes of higher molecular weight. When incubated with Small Unilamellar Vesicles (SUVs) formed with complex lipids, both native and recombinant GRA2 were shown to associate with the SUV membranes and to induce the formation of long membranous tubules observed by transmission electron microscopy (TEM). In the literature, it has been reported that most proteins capable of inducing membrane curvature, interact with phosphatidylinositol (4,5) bi-phosphate (PI(4,5)P2). Fat blots and binding assays of GRA2 to SUVs of de ned lipid composition and containing PI(4,5)P2 showed that GRA2 recognizes phosphoinositides and associates preferentially with PI(4,5)P2-containing SUVs. TEM showed that GRA2 deforms membranes only when PI(4,5)P2 is present. Together, these results allowed us to propose a model of membrane deformation induced by GRA2, the principal effector of the MNN formation.

Biochemical and Biophysical Research Communications, 2015

The most prominent structural feature of the parasitophorous vacuole (PV) in which the intracellu... more The most prominent structural feature of the parasitophorous vacuole (PV) in which the intracellular parasite Toxoplasma gondii proliferates is a membranous nanotubular network (MNN), which interconnects the parasites and the PV membrane. The MNN function remains unclear. The GRA2 and GRA6 proteins secreted from the parasite dense granules into the PV have been implicated in the MNN biogenesis. Amphipathic alpha-helices (AAHs) predicted in GRA2 and an alpha-helical hydrophobic domain predicted in GRA6 have been proposed to be responsible for their membrane association, thereby potentially molding the MMN in its structure. Here we report an analysis of the recombinant proteins (expressed in detergent-free conditions) by circular dichroism, which showed that full length GRA2 displays an alpha-helical secondary structure while recombinant GRA6 and GRA2 truncated of its AAHs are mainly random coiled. Dynamic light scattering and transmission electron microscopy showed that recombinant GRA6 and truncated GRA2 constitute a homogenous population of small particles (6-8 nm in diameter) while recombinant GRA2 corresponds to 2 populations of particles (∼8-15 nm and up to 40 nm in diameter, respectively). The unusual properties of GRA2 due to its AAHs are discussed.

Veterinary Parasitology, 2011

The protozoan parasite Toxoplasma (T.) gondii is one of the most common zoonotic infectious agent... more The protozoan parasite Toxoplasma (T.) gondii is one of the most common zoonotic infectious agents worldwide. Besides its sexual reproduction in cats, T. gondii can also infect a wide spectrum of other warm-blooded animals. These include animals used for human consumption such as pigs or chickens. Nevertheless, the role of turkeys for the epidemiology of T. gondii infections has not been studied thoroughly. We have established a kinetic ELISA (KELA) for the detection of T. gondii-specific IgG antibodies in turkey serum samples. The test is based on the recombinant dense granule antigens GRA7 and GRA8. These proteins were used as an antigen mixture at a concentration of 0.13 g per well. The overall sensitivity of the assay was between 92.6% and 100% and the specificity ranged from 78.1% to 100%, depending on the method used to calculate these parameters. Using this KELA we examined 1913 turkey serum samples from 14 turkey farms from different areas of Germany. From these sera, 387 produced a signal in the KELA, corresponding to a true seroprevalence of up to 20.2%. The seropositivity rate in individual fattening cycles at individual farms ranged from 0.0% to 77.1%, whereas the rates were highly variable within the individual farms and individual fattening cycles. Consequently, conditions of animal husbandry could not be associated with particular seroprevalence rates. Although seropositivity cannot be linked directly to infectious tissue cysts in the muscle tissue of commercially produced turkey meat, we state that there is a potential risk of being infected by consuming turkey meat products that were not heat treated.

International Journal for Parasitology, 2009

The intracellular protozoan parasite Toxoplasma gondii develops within the parasitophorous vacuol... more The intracellular protozoan parasite Toxoplasma gondii develops within the parasitophorous vacuole (PV), an intracellular niche in which it secretes proteins from secretory organelles named dense granules and rhoptries. Here, we describe a new dense granule protein that should now be referred to as GRA12, and that displays no homology with other proteins. Immunofluorescence and immuno-electron microscopy showed that GRA12 behaves similarly to both GRA2 and GRA6. It is secreted into the PV from the anterior pole of the parasite soon after the beginning of invasion, transits to the posterior invaginated pocket of the parasite where a membranous tubulovesicular network is first assembled, and finally resides throughout the vacuolar space, associated with the mature membranous nanotubular network. GRA12 fails to localise at the parasite posterior end in the absence of GRA2. Within the vacuolar space, like the other GRA proteins, GRA12 exists in both a soluble and a membrane-associated form. Using affinity chromatography experiments, we showed that in both the parasite and the PV soluble fractions, GRA12 is purified with the complex of GRA proteins associated with a tagged version of GRA2 and that this association is lost in the PV membranous fraction.

International Journal for Parasitology, 2010

How eukaryotic pathogens export and sort membrane-bound proteins destined for host-cell compartme... more How eukaryotic pathogens export and sort membrane-bound proteins destined for host-cell compartments is still poorly understood. The dense granules of the intracellular protozoan Toxoplasma gondii constitute an unusual secretory pathway that allows soluble export of the GRA proteins which become membrane-associated within the parasite replicative vacuole. This process relies on both the segregation of the proteins routed to the dense granules from those destined to the parasite plasma membrane and on the sorting of the secreted GRA proteins to their proper final membranous system. Here, we provide evidence that the soluble trafficking of GRA6 to the dense granules relies on the N-terminal domain of the protein, which is sufficient to prevent GRA6 targeting to the parasite plasma membrane. We also show that the GRA6 N-terminal domain, possibly by interacting with negatively charged lipids, is fundamental for proper GRA6 association with the vacuolar membranous network of nanotubes. These results support our emerging model: sorting of transmembrane GRA proteins to the host cell vacuole is mainly driven by the dual role of their N-terminal hydrophilic domain and is compartmentally regulated.