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Pep29 localized at the NT region of the GRA5 protein triggered in vitro CD34-DC migration. (A) Amino acid sequences of the peptides chemically synthesized from the NT region (26 –74) of the PRU GRA5. The common name of each peptide, their number in amino acids, and their migration indexes are indicated. (B) Effect of diluting Pep22 (negative control), Pep27, or of Pep29 on the in vitro migration of CD34-DCs toward CCL19. Results obtained with Pep29 represent the mean percent ( Ϯ sd ) of DCs that migrated, obtained from four experiments performed with DCs from different donors and from one experiment for Pep22 and Pep27.
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Pep29, a peptide derived from the Toxoplasma GRA5 protein, is responsible for human dendritic cellsˈ migration toward the CCR7 ligand.
The migration of DCs is a critical function, enabling information to be carried to where the immunological response occurs. Parasites are known to weaken host immunity by interfering with the functions of DCs and th...
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Context 1
... determine the minimal number of amino acids from the rGRA5 NT region required for an optimal in vitro migra- tory activity, a set of synthetic peptides overlapping the re- gion 26 -74 of the type II PRU GRA5 protein sequence [28] was engineered and tested in vitro (Fig. 4A). The PRU GRA5 sequence was chosen, as PRU ESAs induce better mi- gration of CD34-DC than ESAs recovered from the type I RH parasite strain [15,16]. However, this latter strain was particularly suited to genetic manipulation to construct par- asite GRA KOs and for producing rGRA proteins in E. coli [29]. The acidic Pep29, which ...
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... of CD34-DC than ESAs recovered from the type I RH parasite strain [15,16]. However, this latter strain was particularly suited to genetic manipulation to construct par- asite GRA KOs and for producing rGRA proteins in E. coli [29]. The acidic Pep29, which encompasses aa 30 -58 (re- ferred to as Pep29), provided the highest migration index (1.85; Fig. 4A). The peptides Pep27-Pep38 also displayed significant migratory activities. The other peptides produced by the same process, i.e., Pep19 and Pep27 or Pep22 and Pep29, presented very different activities. As these peptides were produced under exactly the same conditions and puri- fied at the same time on the same column and with the ...
Context 3
... The regions bordering aa 45-58 may thus contribute to the migratory activity, possibly by increasing the number of charges and/or by stabilizing this region. Serial dilutions of Pep27 and Pep29 also showed that 5 g/mL was required to obtain a plateau of in vitro migration indexes, whereas the control Pep22 had no effect at any concentration (Fig. 4B). Based on these results, a Pep29 concentration of 10 g/mL was used in the func- tional experiments reported ...
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Introduction: Atopic dermatitis (AD) is a common allergic eczema that affects up to 10% of adults in developed countries. Immune cells in the epidermis, namely, Langerhans cells (LCs), contribute to the pathogenesis of AD, although their exact role(s) in disease remain unclear.
Methods: We performed immunostaining on human skin and peripheral blood...
Citations
... It states that parasites take advantage of immune cell mobility, particularly dendritic cells (DCs), by invading them. Even more surprisingly, infected cells exhibit a modified phenotype, being more mobile under the influence of parasite-derived proteins, such as GRA5 [14]. This mechanism would allow parasites to be disseminated along lymphatic vessels. ...
Infections with the protozoan parasite Toxoplasma gondii are frequent, but one of its main consequences, ocular toxoplasmosis (OT), remains poorly understood. While its clinical description has recently attracted more attention and publications, the underlying pathophysiological mechanisms are only sparsely elucidated, which is partly due to the inherent difficulties to establish relevant animal models. Furthermore, the particularities of the ocular environment explain why the abundant knowledge on systemic toxoplasmosis cannot be just transferred to the ocular situation. However, studies undertaken in mouse models have revealed a central role of interferon gamma (IFNγ) and, more surprisingly, interleukin 17 (IL17), in ocular pathology and parasite control. These studies also show the importance of the genetic background of the infective Toxoplasma strain. Indeed, infections due to exotic strains show a completely different pathophysiology, which translates in a different clinical outcome. These elements should lead to more individualized therapy. Furthermore, the recent advance in understanding the immune response during OT paved the way to new research leads, involving immune pathways poorly studied in this particular setting, such as type I and type III interferons. In any case, deeper knowledge of the mechanisms of this pathology is needed to establish new, more targeted treatment schemes.
... Nonetheless, parasite effectors that stimulate the migratory ability of DCs need to be characterized. TgGRA5, secreted and inserted at the PVM during parasite intracellular replication, was identified as a factor stimulating migration of human DCs in vitro [99]. The authors further pinpointed a peptide derived from the type I-TgGRA5 hydrophilic N-terminal region, which, after internalization by micropinocytosis, triggers the expression of CCR7 and enhances DC directional migration via a JNK-dependent signaling [99]. ...
... TgGRA5, secreted and inserted at the PVM during parasite intracellular replication, was identified as a factor stimulating migration of human DCs in vitro [99]. The authors further pinpointed a peptide derived from the type I-TgGRA5 hydrophilic N-terminal region, which, after internalization by micropinocytosis, triggers the expression of CCR7 and enhances DC directional migration via a JNK-dependent signaling [99]. However, how this peptide acts at the PVM during infection by live parasites remains to be determined. ...
Toxoplasma gondii (T. gondii) est un parasite intracellulaire obligatoire responsable d’une zoonose cosmopolite, la toxoplasmose. Chez les sujets sains, les parasites de type II persistent sous forme de kystes cérébraux, responsables de maladies mentales sévères et augmentant le risque d’apparition de maladies neurodégénératives. Le contrôle de la toxoplasmose chronique dépend de la sécrétion de l’interleukine pro-inflammatoire IL-12 par les cellules dendritiques (DCs) permettant l’activation des lymphocytes T CD8+ (LTs CD8+) cytotoxiques et la sécrétion de l’IFN-g. Les LTs CD8+ jouent un rôle clé dans l’élimination des cellules parasitées durant la phase aigüe de l’infection, mais aussi dans l’établissement d’une immunité protectrice à long terme. Afin de survivre et persister, T. gondii sécrète de nombreux facteurs de virulence qui modulent les réponses immunitaires de son hôte.Des travaux récents ont mis en évidence le rôle clé de la réponse UPR (Unfolded Protein Response) dans la régulation des réponses immunitaires. La réponse UPR est une réponse cytoprotectrice induite durant un stress cellulaire déclenché lors de variations dans l’homéostasie protéique et lipidique au sein du réticulum endoplasmique mais aussi lors d’un stress infectieux. Jusqu’à présent, l’influence de l’infection par T. gondii sur la réponse UPR n’est pas connue. Nous avons émis l’hypothèse que l’induction de l’UPR par T. gondii dans les DCs pourrait moduler leur capacité de présentation antigénique et la sécrétion de cytokines inflammatoires, impactant ainsi la dissémination et la persistance du parasite.En utilisant, des DCs déficientes pour certains effecteurs de l’UPR (IRE1a et XBP1s), nous avons examiné l’impact de leur activité sur les DCs infectées. In vitro, nos résultats ont montré que T. gondii active la voie IRE1a de l’UPR d’une manière dépendante de la voieMyD88 et contrôle ainsi la production des cytokines pro-inflammatoires IL-6 et IL-12 et la présentation antigénique d’antigènes parasitaires par le CMH-I. Dans les souris infectées,la voie IRE1a est spécifiquement activée dans la sous-population de cDC1 et régule l’activation des LTs CD8+, essentiels à la production de l’IFN-g. De plus, les souris déficientes pour IRE1a et XBP1 spécifiquement dans les DCs ne contrôlent pas la prolifération des parasites et succombent à l’infection aigüe. Notre étude révèle donc un rôle protecteur essentiel de cette voie dans les DCs pour combattre l’infection à T. gondii.
... Nonetheless, parasite effectors that stimulate the migratory ability of DCs need to be characterized. TgGRA5, secreted and inserted at the PVM during parasite intracellular replication, was identified as a factor stimulating migration of human DCs in vitro [99]. The authors further pinpointed a peptide derived from the type I-TgGRA5 hydrophilic N-terminal region, which, after internalization by micropinocytosis, triggers the expression of CCR7 and enhances DC directional migration via a JNK-dependent signaling [99]. ...
... TgGRA5, secreted and inserted at the PVM during parasite intracellular replication, was identified as a factor stimulating migration of human DCs in vitro [99]. The authors further pinpointed a peptide derived from the type I-TgGRA5 hydrophilic N-terminal region, which, after internalization by micropinocytosis, triggers the expression of CCR7 and enhances DC directional migration via a JNK-dependent signaling [99]. However, how this peptide acts at the PVM during infection by live parasites remains to be determined. ...
Toxoplasma gondii (Tg), an obligate intracellular parasite of the phylum Apicomplexa, infects a wide range of animals, including humans. A hallmark of Tg infection is the subversion of host responses, which is thought to favor parasite persistence and propagation to new hosts. Recently, a variety of parasite-secreted modulatory effectors have been uncovered in fibroblasts and macrophages, but the specific interplay between Tg and dendritic cells (DCs) is just beginning to emerge. In this review, we summarize the current knowledge on Tg-DC interactions, including innate recognition, cytokine production, and antigen presentation, and discuss open questions regarding how Tg-secreted effectors may shape DC functions to perturb innate and adaptive immunity.
... Yet, hypermigratory DCs down-regulate CCR5 and up-regulate CCR7 upon Toxoplasma challenge (Fuks et al., 2012;Weidner et al., 2013), in line with reported chemotactic responses by CD34 + DCs (Diana et al., 2004). Secretion of the dense granule protein GRA5 has been associated with the upregulation of CCR7, and CCR7/CCL19-driven chemotaxis (Persat et al., 2012). Indeed, DCs challenged with soluble GRA5, or live tachyzoites, chemotaxed in a CCL19 gradient (Fuks et al., 2012;Persat et al., 2012;Weidner et al., 2013). ...
... Secretion of the dense granule protein GRA5 has been associated with the upregulation of CCR7, and CCR7/CCL19-driven chemotaxis (Persat et al., 2012). Indeed, DCs challenged with soluble GRA5, or live tachyzoites, chemotaxed in a CCL19 gradient (Fuks et al., 2012;Persat et al., 2012;Weidner et al., 2013). ...
Dendritic cells (DCs) are regarded as the gatekeepers of the immune system but can also mediate systemic dissemination of the obligate intracellular parasite Toxoplasma gondii. Here, we review the current knowledge on how T. gondii hijacks the migratory machinery of DCs and microglia. Shortly after active invasion by the parasite, infected cells synthesize and secrete the neurotransmitter γ-aminobutyric acid (GABA) and activate GABA-A receptors, which sets on a hypermigratory phenotype in parasitized DCs in vitro and in vivo. The signaling molecule calcium plays a central role for this migratory activation as signal transduction following GABAergic activation is mediated via the L-type voltage-dependent calcium channel (L-VDCC) subtype Cav1.3. These studies have revealed that DCs possess a GABA/L-VDCC/Cav1.3 motogenic signaling axis that triggers migratory activation upon T. gondii infection. Moreover, GABAergic migration can cooperate with chemotactic responses. Additionally, the parasite-derived protein Tg14-3-3 has been associated with hypermigration of DCs and microglia. We discuss the interference of T. gondii infection with host cell signaling pathways that regulate migration. Altogether, T. gondii hijacks non-canonical signaling pathways in infected immune cells to modulate their migratory properties, and thereby promote its own dissemination.
... The introduction of purified Tg14-3-3 into DCs or the expression of Tg14-3-3 in DCs is sufficient to induce hypermotility (54). In addition, a peptide from the T. gondii dense granule protein GRA5 increases the CCR7-mediated chemotaxis of DCs (55). There is also evidence that soluble T. gondii proteins can affect immune cell chemotaxis, as cyclophilin-18 (C-18), a soluble protein secreted by T. gondii, recruits DCs (56) in a CCR5-dependent manner. ...
The motility of blood monocytes is orchestrated by the activity of cell surface integrins, which translate extracellular signals into cytoskeletal changes to mediate adhesion and migration. Toxoplasma gondii is an intracellular parasite that infects migratory cells and enhances their motility, but the mechanisms underlying T. gondii-induced hypermotility are incompletely understood. We have investigated the molecular basis for the hypermotility of primary human peripheral blood monocytes and THP-1 cells infected with T. gondii. Compared to uninfected monocytes, T. gondii infection of monocytes reduced cell spreading and the number of activated β1 integrin clusters in contact with fibronectin during settling, an effect not observed in monocytes treated with LPS or E. coli. Furthermore, T. gondii infection disrupted the phosphorylation of focal adhesion kinase (FAK) at tyrosine 397 (Y397) and Y925 and of the related protein proline-rich tyrosine kinase (Pyk2) at Y402. The localization of paxillin, FAK, and vinculin to focal adhesions and the colocalization of these proteins with activated β1 integrins were also impaired in T. gondii-infected monocytes. Using time-lapse confocal microscopy of THP-1 cells expressing eGFP-FAK during settling on fibronectin, we found that T. gondii-induced monocyte hypermotility was characterized by a reduced number of eGFP-FAK-containing clusters over time compared to uninfected cells. This study demonstrates an integrin conformation-independent regulation of the β1 integrin adhesion pathway, providing further insight into the molecular mechanism of T. gondii- induced monocyte hypermotility.
... Indeed quantification of the antigen expression levels in extracellular tachyzoites using the anti-HF10 antibody, revealed that GRA5-HF10 was at least as abundant as GRA6-HF10 (Fig. 4D). Previous studies reported that GRA5 and GRA6 may directly modulate the function of innate immune cells [33,34], which could affect the T cell stimulation ability of infected BMMs independently of the intrinsic antigenicity of GRA5-HF10 and GRA6-HF10. To evaluate this potential confounding factor, we measured presentation of the exogenously pulsed peptide SM9 thanks to BDSM9Z reporter hybridomas [27]. ...
The intracellular Toxoplasma gondii parasite replicates within a parasitophorous vacuole (PV). T. gondii secretes proteins that remain soluble in the PV space, are inserted into PV membranes or are exported beyond the PV boundary. In addition to supporting T. gondii growth, these proteins can be processed and presented by MHC I for CD8(+) T-cell recognition. Yet it is unclear whether membrane binding influences the processing pathways employed and if topology of membrane antigens impacts their MHC I presentation. Here we report that the MHC I pathways of soluble and membrane-bound antigens differ in their requirement for host ER recruitment. In contrast to the soluble SAG1-OVA model antigen, we find that presentation of the membrane-bound GRA6 is independent from the SNARE Sec 22b, a key molecule for transfer of host endoplasmic reticulum components onto the PV. Using parasites modified to secrete a transmembrane antigen with opposite orientations, we further show that MHC I presentation is highly favored when the C-terminal epitope is exposed to the host cell cytosol, which corresponds to GRA6 natural orientation. Our data suggest that the biochemical properties of antigens released by intracellular pathogens critically guide their processing pathway and are valuable parameters to consider for vaccination strategies. This article is protected by copyright. All rights reserved.
... Polymorphic type I GRA6 was recently shown to manipulate the host cell by activating the host transcription factor nuclear factor of activated T cells 4 (NFAT4) [35]. GRA5 increases the expression of CCR7 [36] and GRA25 induces the expression of CCL2 and C-X-C motif ligand 1 (CXCL1) [37]. Other GRA proteins are exported from the PV into the host cell cytosol and/or nucleus where they modify host cell signaling pathways [38]. ...
Toxoplasma gondii actively invades host cells and establishes a parasitophorous vacuole (PV) that accumulates many proteins secreted by the dense granules (GRA proteins). To date, at least 23 GRA proteins have been reported, though the function(s) of most of these proteins still remains unknown. We targeted gene knockouts at ten GRA gene loci (GRA1-10) to investigate the cellular roles and essentiality of these classical GRA proteins during acute infection in the virulent type I RH strain. While eight of these genes (GRA2-9) were successfully knocked out, targeted knockouts at the GRA1 and GRA10 loci were not obtained, suggesting these GRA proteins may be essential. As expected, the Δgra2 and Δgra6 knockouts failed to form an intravacuolar network (IVN). Surprisingly, Δgra7 exhibited hyper-formation of the IVN in both normal and lipid-free growth conditions. No morphological alterations were identified in parasite or PV structures in the Δgra3, Δgra4, Δgra5, Δgra8, or Δgra9 knockouts. With the exception of the Δgra3 and Δgra8 knockouts, all of the GRA knockouts exhibited defects in their infection rate in vitro. While the single GRA knockouts did not exhibit reduced replication rates in vitro, replication rate defects were observed in three double GRA knockout strains (Δgra4Δgra6, Δgra3Δgra5 and Δgra3Δgra7). However, the virulence of single or double GRA knockout strains in CD1 mice was not affected. Collectively, our results suggest that while the eight individual GRA proteins investigated in this study (GRA2-9) are not essential, several GRA proteins may provide redundant and potentially important functions during acute infection.
... Le CRC co-localise avec la Rétromère. La première fonction connue pour les protéines de granules denses est la maturation de la VP, avec la mise en place du réseau sous-vacuolaire, le passage de molécules entre la cellule et le parasite (Persat et al., 2012) ; (Cesbron-Delauw et al., 2008) ; (Gold et al., 2015). Récemment, plusieurs travaux démontrent aussi l'implication des protéines de granules dense dans la modulation des voies de signalisation de la cellule hôte (Rosowski et al., 2011). ...
... modulation des gènes de la cellule hôte(Persat et al., 2012) ;(Cesbron-Delauw et al., 2008) ;(Gold et al., 2015). Aujourd'hui la question se pose si il existe plusieurs populations différentes de protéines GRA(Mercier and Cesbron- Delauw, 2015). ...
Toxoplasma gondii, comme Plasmodium falciparum appartiennent au phylum des Apicomplexes. Ce groupe de parasites ont comme dénominateurs communs, trois organites apicaux : rhoptries, micronèmes et granules denses contenant des facteurs indispensables pour la reconnaissance, l’entrée et la survie du parasite au sein de la cellule hôte. Le récepteur transmembranaire de type 1 appelé TgSORTLR ("Toxoplasma gondii Sortilin-Like Receptor") est nécessaire à la biogenèse des organelles de sécrétion rhoptrie et micronème (Sloves et al., 2012). Le domaine C-terminale de la TgSORTLR, lie TgVps26 et TgVps35 deux protéines appartement au complexe Rétromère essentiel au recyclage protéique chez les mammifères et S. cerevisiae. Nous avons construit le premier interactome du CRC de T. gondii et des autres Apicomplexes. Contrairement aux mammifères, le Rétromère de T. gondii est composé du CRC (Complexe de Reconnaissance du Cargo) TgVps35-TgVps26-TgVsp29 et l’absence du dimère de Sorting Nexin (SNX). Nous avons identifié plusieurs protéines connues de l’ELC (Endosomal-like compartment) ainsi que des protéines parasitaires spécifiques. La déplétion conditionnelle de TgVps35 démontre que le complexe Rétromère n’est pas seulement crucial pour la biogenèse des rhoptries, micronèmes et granules denses, mais aussi pour l’architecture et l’intégrité du parasite. Nous avons montré que le recyclage de la TgSORTLR entre l’ELC et le TGN (Tans-Golgi-Network) est essentiel au trafic des protéines de sécrétion rhoptries et micronèmes. Par ailleurs nous avons décrit deux nouvelles protéines hypothétiques TgHP12 et TgHP03 pouvant être impliquées respectivement dans le trafic vers l’ELC et vers la membrane plasmique. Afin nous avons identifié et caractérisé une protéine chimérique TgHP25 avec les domaines BAR et SBF2, pouvant être impliquée dans la biogenèse de l’organite rhoptrie. En somme notre travail souligne l’importance du recyclage protéique et l’implication de protéines spécifiques dans la maturation des organites et l’intégrité du parasite.
... In summary, from the several functions defined for the 'canonical' GRA proteins at the tachyzoite stage and from their persistent expression in the cyst wall, it appears that these proteins would play major structural functions within the PV and later in the cyst wall. Moreover, their particular location within the PV at the interface between the host cell and the parasite allows them to be involved in various interactions with the host cell [26,[33][34][35][36][37][38][39]. ...
... Markedly, short-ASP5 is not detectable when a cDNA copy of the gene is expressed in the parasites (Fig 1B and 1C). To determine if the short-ASP5 form identified by western blot is the result of a processing event, a pulse-chase experiment followed by co-immunoprecipitation (IP) was performed using anti-Ty antibodies on 35 S-methionine metabolically labelled ASP5-3Ty expressing parasites. Even during the short pulse, short-ASP5 is readily detectable suggesting that it originates either from an alternative transcriptional initiation, splicing or translational start, but not from a processing maturation event (S1A Fig). ...
... Specifically, upon infection by tachyzoites, DCs exhibit a hypermigratory phenotype [33,34]. GRA5 has been described as one of the parasite effector molecules capable of increasing the migratory properties of DCs via CCR7 expression without DC activation [35]. To determine the potential impact of ASP5 in this process we first assessed the hypermotility phenotype and observed no significant differences between Δasp5 and the corresponding parental lines either in RH or PRU parasites (Fig 7A). ...
... This step is critical for the establishment of infection and persistence as it gives the parasite time to reach sanctuary organs prior to the onset of the immune response [47]. The mediators of DCs hypermigration are not fully characterized; however GRA5 has been reported to be associated with this phenomenon [35]. Parasites lacking ASP5 show a considerable loss of this capacity, which correlates with the down-regulation of CCR7 observed in the RNA seq data upon Δasp5 type I and type II parasite infection. ...
Author Summary
The opportunistic pathogen Toxoplasma gondii infects a large range of nucleated cells where it replicates intracellularly within a parasitophorous vacuole (PV) surrounded by a membrane (PVM). Parasites constitutively secrete dense-granule proteins (GRAs) both into and beyond the PV which participate in remodelling of the PVM, recruitment of host organelles, neutralization of the host cellular defences, and subversion of host cell functioning. In addition, the GRAs critically contribute to cyst wall formation, a process that critically ensures parasite persistence and transmission. To act as effector molecules, some of the GRAs must be translocated across the PVM. Within the related apicomplexan parasite P. falciparum, a repertoire of proteins exported beyond the PVM contain a motif cleaved by a specific protease, Plasmepsin V. Examination of the repertoire of GRAs in T. gondii revealed that some proteins exhibit such export-like motifs suggestive of protease involvement. In this study, we have functionally characterized the related aspartyl protease 5 (TgASP5) in both virulent and persistent T. gondii strains, and have investigated the phenotypic consequences of its deletion in the context of overall parasite biology, its intracellular niche, the infected host cells and the murine model. Our findings revealed fundamental roles of TgASP5 at the host-parasite interface.
