In Silico Identification of Specialized Secretory-Organelle Proteins in Apicomplexan Parasites and In Vivo Validation in Toxoplasma gondii

Apicomplexan parasites, including the human pathogens Toxoplasma gondii and Plasmodium falciparum, employ specialized secretory organelles (micronemes, rhoptries, dense granules) to invade and survive within host cells. Because molecules secreted from these organelles function at the host/parasite interface, their identification is important for understanding invasion mechanisms, and central to the development of therapeutic strategies. Using a computational approach based on predicted functional domains, we have identified more than 600 candidate secretory organelle proteins in twelve apicomplexan parasites. Expression in transgenic T. gondii of eight proteins identified in silico confirms that all enter into the secretory pathway, and seven target to apical organelles associated with invasion. An in silico approach intended to identify possible host interacting proteins yields a dataset enriched in secretory/transmembrane proteins, including most of the antigens known to be engaged by apicomplexan parasites during infection. These domain pattern and projected interactome approaches significantly expand the repertoire of proteins that may be involved in host parasite interactions

Application de la résonance magnétique nucléaire en radioprotection: évolution des temps de relaxation T

L’irradiation totale de la souris au 60Co entraîne une importante diminution des temps de relaxation longitudinale (T1) et transversale (T2) des protons de l’eau tissulaire de la rate par comparaison avec les valeurs obtenues chez les animaux témoins non irradiés. L’expérimentation effectuée par résonance magnétique nucléaire (RMN) pour des doses comprises entre 0,5 Gy et 8 Gy montre que cette diminution est précoce: elle intervient dès le premier jour suivant l'irradiation et persiste jusqu’au cinquième jour. Le retour aux valeurs normales apparaît vers le quinzième jour. L'étude des variations relatives des temps de relaxation fait ressortir une relation linéaire effet-dose qui peut être déterminée in vivo, dans les imageurs RMN, pour évaluer la dose reçue lors d’irradiations accidentelles ou à visée thérapeutique. Les variations observées au niveau des autres organes étudiés (poumon, rein, foie, cerveau) sont par contre très faibles et peu significatives

High spatial resolution quantitative MR images : an experimental study of dedicated surface poils.

International audienc

Sub-localisation of novel proteins with unique dynamics during assembly and maintenance of the eukaryotic flagellum

International audienceCilia and flagella are complex organelles composed of up to 500 proteins. We have purified intact flagella from the model organism Trypanosoma brucei using mechanical shearing. Scanning and transmission electron microscopy confirmed the quality and the purity of flagella and biochemical analysis demonstrated a 15-fold enrichment of flagellar markers. Mass spectrometry investigation carried out on 5 separate experiments led to the identification of 387 proteins, 55 of which had never reported to be associated to the flagellum. 10 out of the 12 proteins investigated experimentally were indeed associated to the flagellum but turned out to localise to several sub-localisations with unique profiles: flagellar membrane, axoneme, paraflagellar rod (an extra-axonemal structure) and the adhesion zone. Two of them, termed FLAMM6 and FLAMM8 showed restricted distribution to the proximal part and to the far distal end of the axoneme, respectively. Dynamics analysis revealed that membrane proteins were incorporated by the proximal end and showed a rapid turnover whereas axonemal and PFR proteins were added to the distal end of elongating flagella but showed stable association to their structure. FLAMM6 was found only in the first half of the flagellum no matter its length, a process dependent on IFT. Finally, FLAMM8 was progressively incorporated to the elongating axoneme accumulating at the distal tip where it showed very slow turnover after flagellum formation was complete. These data highlight the existence of specific micro-domains within the eukaryotic flagellum, each with its own dynamics for assembly and turnover

Dual stage synthesis and crucial role of cytoadherence-linked asexual gene 9 in the surface expression of malaria parasite var proteins

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family members mediate the adherence of parasite-infected red blood cells (IRBCs) to various host receptors. A previous study has shown that the parasite protein, cytoadherence-linked asexual gene 9 (CLAG9), is also essential for IRBC adherence. However, how CLAG9 influences this process remains unknown. In this study, we show that CLAG9 interacts with VAR2CSA, a PfEMP1 that mediates IRBC adherence to chondroitin 4-sulfate in the placenta. Importantly, our results show that the adherent parasites synthesize CLAG9 at two stages—the early ring and late trophozoite stages. Localization studies revealed that a substantial level of CLAG9 is located mainly at or in close proximity of the IRBC membrane in association with VAR2CSA. Upon treatment of IRBCs with trypsin, a significant amount of CLAG9 (≈150 kDa) was converted into ≈142-kDa polypeptide. Together these data demonstrate that a considerable amount of CLAG9 is embedded in the IRBC membrane such that at least a portion of the polypeptide at either N or C terminus is exposed on the cell surface. In parasites lacking CLAG9, VAR2CSA failed to express on the IRBC surface and was located within the parasite. Based on these findings, we propose that CLAG9 plays a critical role in the trafficking of PfEMP1s onto the IRBC surface. These results have important implications for the development of therapeutics for cerebral, placental, and other cytoadherence-associated malaria illnesses

Trypanosome motion represents an adaptation to the crowded environment of the vertebrate bloodstream

Blood is a remarkable habitat: it is highly viscous, contains a dense packaging of cells and perpetually flows at velocities varying over three orders of magnitude. Only few pathogens endure the harsh physical conditions within the vertebrate bloodstream and prosper despite being constantly attacked by host antibodies. African trypanosomes are strictly extracellular blood parasites, which evade the immune response through a system of antigenic variation and incessant motility. How the flagellates actually swim in blood remains to be elucidated. Here, we show that the mode and dynamics of trypanosome locomotion are a trait of life within a crowded environment. Using high-speed fluorescence microscopy and ordered micro-pillar arrays we show that the parasites mode of motility is adapted to the density of cells in blood. Trypanosomes are pulled forward by the planar beat of the single flagellum. Hydrodynamic flow across the asymmetrically shaped cell body translates into its rotational movement. Importantly, the presence of particles with the shape, size and spacing of blood cells is required and sufficient for trypanosomes to reach maximum forward velocity. If the density of obstacles, however, is further increased to resemble collagen networks or tissue spaces, the parasites reverse their flagellar beat and consequently swim backwards, in this way avoiding getting trapped. In the absence of obstacles, this flagellar beat reversal occurs randomly resulting in irregular waveforms and apparent cell tumbling. Thus, the swimming behavior of trypanosomes is a surprising example of micro-adaptation to life at low Reynolds numbers. For a precise physical interpretation, we compare our high-resolution microscopic data to results from a simulation technique that combines the method of multi-particle collision dynamics with a triangulated surface model. The simulation produces a rotating cell body and a helical swimming path, providing a functioning simulation method for microorganism with a complex swimming strategy