Synergistic role of micronemal proteins in Toxoplasma gondii virulence

Apicomplexan parasites invade cells by a unique mechanism involving discharge of secretory vesicles called micronemes. Microneme proteins (MICs) include transmembrane and soluble proteins expressing different adhesive domains. Although the transmembrane protein TRAP and its homologues are thought to bridge cell surface receptors and the parasite submembranous motor, little is known about the function of other MICs. We have addressed the role of MIC1 and MIC3, two soluble adhesins of Toxoplasma gondii, in invasion and virulence. Single deletion of the MIC1 gene decreased invasion in fibroblasts, whereas MIC3 deletion had no effect either alone or in the mic1KO context. Individual disruption of MIC1 or MIC3 genes slightly reduced virulence in the mouse, whereas doubly depleted parasites were severely impaired in virulence and conferred protection against subsequent challenge. Single substitution of two critical amino acids in the chitin binding–like (CBL) domain of MIC3 abolished MIC3 binding to cells and generated the attenuated virulence phenotype. Our findings identify the CBL domain of MIC3 as a key player in toxoplasmosis and reveal the synergistic role of MICs in virulence, supporting the idea that parasites have evolved multiple ligand–receptor interactions to ensure invasion of different cells types during the course of infection

Mic1-3 Knockout Toxoplasma gondii is a good candidate for a vaccine against T. gondii-induced abortion in sheep

This study assessed the effectiveness of a mutant strain of Toxoplasma gondii (RH strain) lacking the mic1 and mic3 genes (Mic1-3KO) against Toxoplasma abortion in sheep. Ewes were inoculated subcutaneously with 105 Mic1-3KO tachyzoïtes in three independent experiments. Following vaccination, Mic1-3KO induced a mild febrile response and serum IgG antibodies, which persisted throughout the experiments. Tissue cysts formed in the sheep, but were not, under our experimental conditions, infectious when given orally. Ewes were mated two months after vaccination and were orally challenged with the PRU strain of T. gondii at mid-gestation (400 oocysts in Experiments 1 and 2; 100 oocysts in Experiment 3). Challenge of vaccinated pregnant ewes resulted in a slight febrile response, whereas unvaccinated ewes developed a more severe, characteristic febrile response of longer duration. After challenge, all unvaccinated ewes aborted whereas 62%, 91% and 64% (Experiments 1, 2 and 3 respectively) of the lambs from vaccinated ewes were viable, with no clinical signs of infection. Mic1-3KO was as effective as S48, the strain used as a live vaccine for sheep (Toxovax®). A dose of 105 Mic1-3KO tachyzoites was sufficient to induce protection (versus a dose of 2 × 106). Both subcutaneous and intraperitoneal injections were effective. Moreover, preliminary results showed the potential of Mic1-3KO to reduce the development of tissue cysts in lambs born to vaccinated ewes. This study demonstrates that Mic1-3KO is a potent vaccine candidate

A model of the PI cycle reveals the regulating roles of lipid-binding proteins and pitfalls of using mosaic biological data

The phosphatidylinositol (PI) cycle is central to eukaryotic cell signaling. Its complexity, due to the number of reactions and lipid and inositol phosphate intermediates involved makes it difficult to analyze experimentally. Computational modelling approaches are seen as a way forward to elucidate complex biological regulatory mechanisms when this cannot be achieved solely through experimental approaches. Whilst mathematical modelling is well established in informing biological systems, many models are often informed by data sourced from multiple unrelated cell types (mosaic data) or from purified enzyme data. In this work, we develop a model of the PI cycle informed by experimental and omics data taken from a single cell type, namely platelets. We were able to make a number of predictions regarding the regulation of PI cycle enzymes, the importance of the number of receptors required for successful GPCR signaling and the importance of lipid- and protein-binding proteins in regulating second messenger outputs. We then consider how pathway behavior differs, when fully informed by data for HeLa cells and show that model predictions remain consistent. However, when informed by mosaic experimental data model predictions greatly vary illustrating the risks of using mosaic datasets from unrelated cell types

Prothrombotic and Proinflammatory Activities of the β-Hemolytic Group B Streptococcal Pigment

A prominent feature of severe streptococcal infections is the profound inflammatory response that contributes to systemic toxicity. In sepsis the dysregulated host response involves both immunological and nonimmunological pathways. Here, we report a fatal case of an immunocompetent healthy female presenting with toxic shock and purpura fulminans caused by group B streptococcus (GBS; serotype III, CC19). The strain (LUMC16) was pigmented and hyperhemolytic. Stimulation of human primary cells with hyperhemolytic LUMC16 and STSS/NF-HH strains and pigment toxin resulted in a release of proinflammatory mediators, including tumor necrosis factor, interleukin (IL)-1β, and IL-6. In addition, LUMC16 induced blood clotting and showed factor XII activity on its surface, which was linked to the presence of the pigment. The expression of pigment was not linked to a mutation within the CovR/S region. In conclusion, our study shows that the hemolytic lipid toxin contributes to the ability of GBS to cause systemic hyperinflammation and interferes with the coagulation system

Etudes des mécanismes moléculaires de l'invasion des cellules hotes par Toxoplasma gondii (rôle des protéines de micronèmes)

Cell invasion by apicomplexan is associated ith secretion of microneme proteins. We have investigated the role of two soluble MIC1 and MIC3 proteins, which have affinity for cell surface. We have developped an original MIC3 binding essay by transfection of mammalian cells. The receptor binding site of MIC3 is located in the N-terminal chitin binding-like domain, which remains poorly accessible until the adjacent propetide has been cleaved, and shown that binding requires dimerization. Two aromatic amino acids in this domain have been identified to be crucial for binding to cells. Mic1KO, mic1-3KO or mic1-3KO parasites have been genetically engineered. While individual disruption of MIC1 or MIC3 genes slightly reduced virulence, doubly depleted parasites were markedly impaired in virulence and confered protection against oral cyst challenge. Deletion of the MIC1 gene alone affected fibroblast invasion. MIC3 binding function is crucial for expression of virulence.L'invasion des cellules par les Apicomplexa est associée à la sécrétion de protéines de micronèmes. Nous avons étudié la fonction de deux protéines, MIC1 et MIC3, possédant des propriétés d'adhésion pour la surface des cellules in vitro. Le domaine d'adhésion de MIC3 est apparenté au domaine de fixation du N-actétylglucosamine. Il est fonctionnel sous forme dimérique et seulement après clivage d'une proséquence. Deux acides aminés aromatiques indispensables à la fonction de reconnaissance de la cellule hôte ont été identifiés. Après délétion par génétique inverse des gènes MIC1 et MIC3 indépendamment ou simultanément et nous avons montré le rôle additif de ces protéines dans la virulence. Seulement le Mic1KO présente un défaut d'invasion. Le rôle de MIC3 dans la virulence dépend de ses propriétés adhésives pour la surface des cellules hôtes. Des essais de vaccination utilisant le double mutant conduisent à une protection totale des souris vis à vis d'une seconde infection par voie orale.TOURS-BU Sciences Pharmacie (372612104) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Facteurs et mécanismes contribuant à l'infection congénitale à trypanosoma cruzi

TOURS-BU Sciences Pharmacie (372612104) / SudocSudocFranceF

Rôle des cellules dendritiques muqueuses et des exosomes dans la réponse immunitaire protectrice anti-toxoplasma gondii

Toxoplasma gondii est un parasite protozoaire intracellulaire obligatoire, infectant tous les animaux à sang chaud. Généralement bénigne, la toxoplasmose peut revêtir un caractère de grande sévérité chez l'homme, dans les cas d'immunosuppression et de contamination transplacentaire du foetus. La toxoplasmose est un problème de santé publique et justifie l'importance des travaux consacrés à la mise au point d'un vaccin anti-parasitaire. L'hôte immunocompétent, infecté par voie orale, développe une réponse immunitaire protectrice, humorale et cellulaire, à la fois muqueuse et systémique. Depuis quelques années, un intérêt grandissant se porte sur la vaccination par voie muqueuse en raison de la contribution du système immunitaire muqueux au contrôle de la toxoplasmose. Des études suggèrent également un rôle-clef des cellules dendritiques (CD), cellules présentatrices d'antigènes professionnelles, dans la réponse immune vis-à-vis du toxoplasme. C'est pourquoi nous nous sommes intéressés au rôle inducteur mais également au rôle effecteur potentiel des CD dans l'immunité anti-toxoplasmique ainsi qu'aux mécanismes immunitaires mis en jeu et aboutissant à la protection et plus particulièrement au rôle des exosomes dérivés de CD. En ce qui concerne le rôle inducteur des CD, nous avons pu observer que le transfert de CD isolées à partir de ganglions mésentériques ou d'exosomes sensibilisées par des extraits antigéniques totaux de T. gondii, à des animaux receveurs contrôles, confère une forte protection contre une épreuve virulente associée à une réponse lymphoproliférative splénique et locale. Ces résultats montrent le rôle prépondérant des CD et des exosomes qu'elles produisent dans l'initiation d'une réponse immunitaire protectrice anti-T.gondii. Parallèlement, nous nous sommes intéressés au rôle effecteur des CD. Nos résultats démontrent la capacité microbiostatique des CD activées par l'IFN-g et placent désormais les CD comme principaux médiateurs dans le contrôle de l'infection toxoplasmique.TOURS-BU Sciences Pharmacie (372612104) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Vaccination muqueuse contre la toxoplasmose (effet adjuvant de mutants de la toxine lt d'e. Coli sur les réponses immunitaires et la protection)

TOURS-BU Sciences Pharmacie (372612104) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF