Hijacking of the Pleiotropic Cytokine Interferon-γ by the Type III Secretion System of Yersinia pestis

Yersinia pestis, the causative agent of bubonic plague, employs its type III secretion system to inject toxins into target cells, a crucial step in infection establishment. LcrV is an essential component of the T3SS of Yersinia spp, and is able to associate at the tip of the secretion needle and take part in the translocation of anti-host effector proteins into the eukaryotic cell cytoplasm. Upon cell contact, LcrV is also released into the surrounding medium where it has been shown to block the normal inflammatory response, although details of this mechanism have remained elusive. In this work, we reveal a key aspect of the immunomodulatory function of LcrV by showing that it interacts directly and with nanomolar affinity with the inflammatory cytokine IFNγ. In addition, we generate specific IFNγ mutants that show decreased interaction capabilities towards LcrV, enabling us to map the interaction region to two basic C-terminal clusters of IFNγ. Lastly, we show that the LcrV-IFNγ interaction can be disrupted by a number of inhibitors, some of which display nanomolar affinity. This study thus not only identifies novel potential inhibitors that could be developed for the control of Yersinia-induced infection, but also highlights the diversity of the strategies used by Y. pestis to evade the immune system, with the hijacking of pleiotropic cytokines being a long-range mechanism that potentially plays a key role in the severity of plague

Structural Basis of Cytotoxicity Mediated by the Type III Secretion Toxin ExoU from Pseudomonas aeruginosa

The type III secretion system (T3SS) is a complex macromolecular machinery employed by a number of Gram-negative pathogens to inject effectors directly into the cytoplasm of eukaryotic cells. ExoU from the opportunistic pathogen Pseudomonas aeruginosa is one of the most aggressive toxins injected by a T3SS, leading to rapid cell necrosis. Here we report the crystal structure of ExoU in complex with its chaperone, SpcU. ExoU folds into membrane-binding, bridging, and phospholipase domains. SpcU maintains the N-terminus of ExoU in an unfolded state, required for secretion. The phospholipase domain carries an embedded catalytic site whose position within ExoU does not permit direct interaction with the bilayer, which suggests that ExoU must undergo a conformational rearrangement in order to access lipids within the target membrane. The bridging domain connects catalytic domain and membrane-binding domains, the latter of which displays specificity to PI(4,5)P2. Both transfection experiments and infection of eukaryotic cells with ExoU-secreting bacteria show that ExoU ubiquitination results in its co-localization with endosomal markers. This could reflect an attempt of the infected cell to target ExoU for degradation in order to protect itself from its aggressive cytotoxic action

Etude de l'adressage des protéines GRAs transmembranaires de Toxoplasma gondii aux granules denses et de leur insertion membranaire post-sécrétoire.

The success of many intracellular pathogens relies on the export of both soluble and membrane-bound proteins that are destined to modify various compartments of the host cell. In Toxoplasma gondii, it is well established that the dense granules (DG) constitute the default constitutive pathway for soluble proteins. By contrast, the mechanism by which transmembrane proteins are sorted to the DG and are maintained in a soluble state while adopting a transmembrane topology after secretion is not known.The GRA5 DG protein of T. gondii is targeted to the parasitophorous vacuole membrane (PVM) after soluble secretion. Expression of GRA5 in mammalian cells revealed that the protein is targeted to the cell surface with a type I topology, providing evidence that soluble trafficking of GRA5 within the parasite is peculiar. By using chimeric proteins containing specific domains of GRA5 and of a parasite plasma membrane (PPM) targeted transmembrane protein, we investigated which are the determinant(s) of PPM versus PVM targeting. We demonstrated that the GRA5 Nt domain is involved in soluble targeting within the DG and is essential for insertion into the PVM. These results, that were extented to another transmembrane GRA protein (GRA6), contrast with the broad acceptance that sorting signals are present within the cytoplasmic tail of membranous proteins and/or depend on the size of their transmembrane domain.Parmi les mécanismes de survie intracellulaire connus, l'export de protéines solubles ou transmembranaires visant à modifier différents compartiments de la cellule-hôte est une stratégie employée par de nombreux pathogènes. Chez Toxoplasma gondii, il a été montré que les granules denses (GD) constituent la voie de sécrétion par défaut pour les protéines solubles. Par contre, le tri de protéines transmembranaires vers les GD et leur maintien sous forme soluble avant insertion membranaire post-sécrétoire font appel à des mécanismes originaux qui ont fait l'objet de ces travaux.Chez le Toxoplasme, la protéine de GD GRA5 est adressée à la membrane de la vacuole parasitophore (MVP) après sécrétion. Exprimée en cellules de mammifères, GRA5 est adressée à la membrane plasmique avec une topologie de type I, ce qui démontre la particularité des mécanismes de sécrétion chez T. gondii. Par une approche basée sur des protéines chimériques présentant des domaines spécifiques de GRA5 et d'une protéine transmembranaire de la membrane plasmique parasitaire (MPP), nous avons pu identifier les déterminants de l'adressage à la MPP versus à la MVP. Nous avons ainsi pu démontrer que le domaine Nt de GRA5 est impliqué dans l'adressage soluble aux GD et est essentiel pour l'insertion membranaire post-sécrétoire dans la MVP. Ces résultats, qui ont été étendus à une autre protéine GRA transmembranaire (GRA6), divergent de l'idée largement répandue selon laquelle les signaux d'adressage des protéines transmembranaires seraient présents dans la queue C-terminale et/ou dépendraient de la longueur du domaine transmembranaire de ces protéines

Claire Gendrin

Gendrin Claire. Claire Gendrin. In: Diplômées, n°238, 2011. Femmes diplômées de l’enseignement supérieur et temps partiel. pp. 163-164

Etude de l adressage des protéines GRAs transmembranaires de Toxoplasma gondii aux granules denses et de leur insertion membranaire post-sécrétoire

Parmi les mécanismes de survie intracellulaire connus, l export de protéines solubles ou transmembranaires visant à modifier différents compartiments de la cellule-hôte est une stratégie employée par de nombreux pathogènes. Chez Toxoplasma gondii, il a été montré que les granules denses (GD) constituent la voie de sécrétion par défaut pour les protéines solubles. Par contre, le tri de protéines transmembranaires vers les GD et leur maintien sous forme soluble avant insertion membranaire post-sécrétoire font appel à des mécanismes originaux qui ont fait l objet de ces travaux. Chez le Toxoplasme, la protéine de GD GRA5 est adressée à la membrane de la vacuole parasitophore (MVP) après sécrétion. Exprimée en cellules de mammifères, GRA5 est adressée à la membrane plasmique avec une topologie de type I, ce qui démontre la particularité des mécanismes de sécrétion chez T. gondii. Par une approche basée sur des protéines chimériques présentant des domaines spécifiques de GRA5 et d une protéine transmembranaire de la membrane plasmique parasitaire (MPP), nous avons pu identifier les déterminants de l adressage à la MPP versus à la MVP. Nous avons ainsi pu démontrer que le domaine Nt de GRA5 est impliqué dans l adressage soluble aux GD et est essentiel pour l insertion membranaire post-sécrétoire dans la MVP. Ces résultats, qui ont été étendus à une autre protéine GRA transmembranaire (GRA6), divergent de l idée largement répandue selon laquelle les signaux d adressage des protéines transmembranaires seraient présents dans la queue C-terminale et/ou dépendraient de la longueur du domaine transmembranaire de ces protéines.The success of many intracellular pathogens relies on the export of both soluble and membrane-bound proteins that are destined to modify various compartments of the host cell. In Toxoplasma gondii, it is well established that the dense granules (DG) constitute the default constitutive pathway for soluble proteins. By contrast, the mechanism by which transmembrane proteins are sorted to the DG and are maintained in a soluble state while adopting a transmembrane topology after secretion is not known. The GRA5 DG protein of T. gondii is targeted to the parasitophorous vacuole membrane (PVM) after soluble secretion. Expression of GRA5 in mammalian cells revealed that the protein is targeted to the cell surface with a type I topology, providing evidence that soluble trafficking of GRA5 within the parasite is peculiar. By using chimeric proteins containing specific domains of GRA5 and of a parasite plasma membrane (PPM) targeted transmembrane protein, we investigated which are the determinant(s) of PPM versus PVM targeting. We demonstrated that the GRA5 Nt domain is involved in soluble targeting within the DG and is essential for insertion into the PVM. These results, that were extented to another transmembrane GRA protein (GRA6), contrast with the broad acceptance that sorting signals are present within the cytoplasmic tail of membranous proteins and/or depend on the size of their transmembrane domain.GRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF

Post-translational membrane sorting of the Toxoplasma gondii GRA6 protein into the parasite-containing vacuole is driven by its N-terminal domain.

International audienceHow 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

Characterization of the LcrV/IFNγ interaction by intrinsic fluorescence studies.

<p>(A) Effects of NBS modification on LcrV spectral properties. LcrV was incubated with an 80-fold molar excess of NBS, and the resulting fluorescence (grey curve) was compared to that of wild-type LcrV (black curve). The fluorescence emission spectrum was recorded over the range 310–370 nm after excitation at 295 nm. (B) Evolution of the relative fluorescence intensities over injection of increasing concentrations of oxidized LcrV (LcrVox). Fitting of the data was performed as described in the experimental section. The LcrV curve was not shown for clarity. F_IFNγ; intensity of the IFNγ spectrum; F_NATA, intensity of the NATA spectrum.</p

Free IFNγ interacts with LcrV, but not with PcrV, in a surface plasmon resonance assay.

<p>(A) Scatchard analysis of IFNγ binding to LcrV immobilized on a CM3 sensorchip (1150 RU). R_eq, steady state value at equilibrium; C, concentration of injected IFNγ. (B) 5.0 µg/mL IFNγ was injected over LcrV (2700 RU, black curve) and PcrV (1200 RU, grey curve) immobilized on two different lanes of the same sensorchip. (C) Injection over immobilized LcrV (1150 RU) of 2 µg/mL IFNγ (black curve) or of 2 µg/mL IFNγ and an equimolar amount of IFNγR (grey curve). RU =  resonance units. (D) Injection of 1 µg/mL IFNγ alone (black curve) or in combination with LcrV (1 µg/mL of each, grey curve) over immobilized IFNγR (1600 RU). For all experiments, non-specific binding to the sensor chip was subtracted from the raw data.</p

Inhibition of LcrV/IFNγ interaction by different synthetic oligosaccharides.

<p>(A) Formula of the different oligosaccharides used in this study. m, number of disaccharide repeats, n = number of ethylene glycol repeats. Adapted from ref 51. (B) Each inhibitor tested was incubated with IFNγ, and complexes were injected over immobilized LcrV (2000 RU) at 10 µL/min. Non-specific binding to the sensor chip was subtracted for each injection. The percentage of inhibition is represented for three independent experiments, and the standard error of the mean is indicated (bars). The mean response in the absence of inhibitor (0% of inhibition) was equal to 270 RU. (C) IFNγ was pre-incubated with a range of concentrations of 2O₃₂. Each reaction mixture was injected over immobilized LcrV (1150 RU) at 10 µL/min. Non-specific binding to the sensor chip was subtracted for each injection.</p