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Fundamental Roles of the Golgi-Associated Toxoplasma Aspartyl Protease, ASP5, at the Host-Parasite Interface.

Hammoudi PM, Jacot D, Mueller C, Di Cristina M, Dogga SK, Marq JB, Romano J, Tosetti N, Dubrot J, Emre Y, Lunghi M, Coppens I, Yamamoto M, Sojka D, Pino P, Soldati-Favre D - PLoS Pathog. (2015)

Bottom Line: We show that deletion of ASP5 causes a significant loss in parasite fitness in vitro and an altered virulence in vivo.Additionally, hypermigration of dendritic cells and bradyzoite cyst wall formation are impaired, critically impacting on parasite dissemination and persistence.Overall, the absence of ASP5 dramatically compromises the parasite's ability to modulate host signalling pathways and immune responses.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Molecular Medicine, Centre Médical Universitaire, University of Geneva, Geneva, Switzerland.

ABSTRACT
Toxoplasma gondii possesses sets of dense granule proteins (GRAs) that either assemble at, or cross the parasitophorous vacuole membrane (PVM) and exhibit motifs resembling the HT/PEXEL previously identified in a repertoire of exported Plasmodium proteins. Within Plasmodium spp., cleavage of the HT/PEXEL motif by the endoplasmic reticulum-resident protease Plasmepsin V precedes trafficking to and export across the PVM of proteins involved in pathogenicity and host cell remodelling. Here, we have functionally characterized the T. gondii aspartyl protease 5 (ASP5), a Golgi-resident protease that is phylogenetically related to Plasmepsin V. We show that deletion of ASP5 causes a significant loss in parasite fitness in vitro and an altered virulence in vivo. Furthermore, we reveal that ASP5 is necessary for the cleavage of GRA16, GRA19 and GRA20 at the PEXEL-like motif. In the absence of ASP5, the intravacuolar nanotubular network disappears and several GRAs fail to localize to the PVM, while GRA16 and GRA24, both known to be targeted to the host cell nucleus, are retained within the vacuolar space. Additionally, hypermigration of dendritic cells and bradyzoite cyst wall formation are impaired, critically impacting on parasite dissemination and persistence. Overall, the absence of ASP5 dramatically compromises the parasite's ability to modulate host signalling pathways and immune responses.

No MeSH data available.


Related in: MedlinePlus

In the absence of ASP5, PV architecture but not host organelle recruitment is altered.(A) In the absence of ASP5 in type I parasites, GRA1 staining was not affected. Conversely, while GRA2 labelling was still present at the PV, it displayed an altered, punctate signal when compared to wt parasites. More strikingly, in the absence of ASP5, GRA3, and GRA7 remained in the vacuolar space and were no longer inserted into the PVM. α-GAP45 antibodies were used to stain the parasite periphery. All IFAs were performed with PFA/GA fixation for 30 min. Scale bars represent 2 μm. (B) Electron micrographs of the membranous nanotubular network (MNN, black arrows) in type I parasites. Strikingly, parasites lacking ASP5 have either no MNN, or occasionally punctate structures (black asterisk) in the vacuolar space. In contrast, ASP5 deletion did not affect the overall ultrastructure of the parasites, particularly the presence of the dense granules (DG). (C) Electron micrographs of infected host cells (hc) showing host mitochondria (hm) association with the PV of RH and RHΔasp5 parasites (p). Scale bars represent 1 μm.
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ppat.1005211.g002: In the absence of ASP5, PV architecture but not host organelle recruitment is altered.(A) In the absence of ASP5 in type I parasites, GRA1 staining was not affected. Conversely, while GRA2 labelling was still present at the PV, it displayed an altered, punctate signal when compared to wt parasites. More strikingly, in the absence of ASP5, GRA3, and GRA7 remained in the vacuolar space and were no longer inserted into the PVM. α-GAP45 antibodies were used to stain the parasite periphery. All IFAs were performed with PFA/GA fixation for 30 min. Scale bars represent 2 μm. (B) Electron micrographs of the membranous nanotubular network (MNN, black arrows) in type I parasites. Strikingly, parasites lacking ASP5 have either no MNN, or occasionally punctate structures (black asterisk) in the vacuolar space. In contrast, ASP5 deletion did not affect the overall ultrastructure of the parasites, particularly the presence of the dense granules (DG). (C) Electron micrographs of infected host cells (hc) showing host mitochondria (hm) association with the PV of RH and RHΔasp5 parasites (p). Scale bars represent 1 μm.

Mentions: To assess the role of ASP5 in trafficking of the GRAs to the PV and/or the PVM, we first examined the localization of the subset of GRAs for which specific antibodies were available (GRA1, 2, 3 and 7). GRA1 is expressed and secreted into the vacuolar space as a soluble protein that subsequently becomes peripherally associated with the MNN [21]. As shown in Fig 2A, the localization of GRA1, which possesses a putative PEXEL-like motif (RALNK), is not affected by the absence of ASP5. In contrast, GRA2 and GRA3 that are associated with the MNN in wt parasites [13], showed an altered staining pattern in Δasp5 parasites (Fig 2A). Upon strong fixation conditions adapted to visualize proteins accumulated in the vacuolar space, GRA2 is not aggregated and displays instead a punctate staining for around 80% of the PVs observed. Similarly, GRA7 and also GRA3 localization at the PVM was modified in the absence of ASP5, with no PVM staining observed in more than 70% of the vacuoles (Fig 2A). Given that several GRAs involved in MNN formation appeared perturbed in RHΔasp5 parasites, the morphology of the PV was examined by electron microscopy. Whilst the MNN in wt parasites is comprised of elongated nanotubules, a dramatic change of vacuolar space architecture was observed in the absence of ASP5. In contrast to RH parasites, the PV of RHΔasp5 parasites did not exhibit a typical MNN which is usually constituted of many long and intricate tubules. Instead, RHΔasp5 parasites contained vesicles and small tubules sparsely distrubuted throughout the vacuolar space (Figs 2B and S4). This indicates that parasites lacking ASP5 are unable to assemble an elaborated MNN. The PV lumen of RHΔasp5 parasites also appears different to that of parasites depleted in both GRA2 and GRA6 [13].


Fundamental Roles of the Golgi-Associated Toxoplasma Aspartyl Protease, ASP5, at the Host-Parasite Interface.

Hammoudi PM, Jacot D, Mueller C, Di Cristina M, Dogga SK, Marq JB, Romano J, Tosetti N, Dubrot J, Emre Y, Lunghi M, Coppens I, Yamamoto M, Sojka D, Pino P, Soldati-Favre D - PLoS Pathog. (2015)

In the absence of ASP5, PV architecture but not host organelle recruitment is altered.(A) In the absence of ASP5 in type I parasites, GRA1 staining was not affected. Conversely, while GRA2 labelling was still present at the PV, it displayed an altered, punctate signal when compared to wt parasites. More strikingly, in the absence of ASP5, GRA3, and GRA7 remained in the vacuolar space and were no longer inserted into the PVM. α-GAP45 antibodies were used to stain the parasite periphery. All IFAs were performed with PFA/GA fixation for 30 min. Scale bars represent 2 μm. (B) Electron micrographs of the membranous nanotubular network (MNN, black arrows) in type I parasites. Strikingly, parasites lacking ASP5 have either no MNN, or occasionally punctate structures (black asterisk) in the vacuolar space. In contrast, ASP5 deletion did not affect the overall ultrastructure of the parasites, particularly the presence of the dense granules (DG). (C) Electron micrographs of infected host cells (hc) showing host mitochondria (hm) association with the PV of RH and RHΔasp5 parasites (p). Scale bars represent 1 μm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4608785&req=5

ppat.1005211.g002: In the absence of ASP5, PV architecture but not host organelle recruitment is altered.(A) In the absence of ASP5 in type I parasites, GRA1 staining was not affected. Conversely, while GRA2 labelling was still present at the PV, it displayed an altered, punctate signal when compared to wt parasites. More strikingly, in the absence of ASP5, GRA3, and GRA7 remained in the vacuolar space and were no longer inserted into the PVM. α-GAP45 antibodies were used to stain the parasite periphery. All IFAs were performed with PFA/GA fixation for 30 min. Scale bars represent 2 μm. (B) Electron micrographs of the membranous nanotubular network (MNN, black arrows) in type I parasites. Strikingly, parasites lacking ASP5 have either no MNN, or occasionally punctate structures (black asterisk) in the vacuolar space. In contrast, ASP5 deletion did not affect the overall ultrastructure of the parasites, particularly the presence of the dense granules (DG). (C) Electron micrographs of infected host cells (hc) showing host mitochondria (hm) association with the PV of RH and RHΔasp5 parasites (p). Scale bars represent 1 μm.
Mentions: To assess the role of ASP5 in trafficking of the GRAs to the PV and/or the PVM, we first examined the localization of the subset of GRAs for which specific antibodies were available (GRA1, 2, 3 and 7). GRA1 is expressed and secreted into the vacuolar space as a soluble protein that subsequently becomes peripherally associated with the MNN [21]. As shown in Fig 2A, the localization of GRA1, which possesses a putative PEXEL-like motif (RALNK), is not affected by the absence of ASP5. In contrast, GRA2 and GRA3 that are associated with the MNN in wt parasites [13], showed an altered staining pattern in Δasp5 parasites (Fig 2A). Upon strong fixation conditions adapted to visualize proteins accumulated in the vacuolar space, GRA2 is not aggregated and displays instead a punctate staining for around 80% of the PVs observed. Similarly, GRA7 and also GRA3 localization at the PVM was modified in the absence of ASP5, with no PVM staining observed in more than 70% of the vacuoles (Fig 2A). Given that several GRAs involved in MNN formation appeared perturbed in RHΔasp5 parasites, the morphology of the PV was examined by electron microscopy. Whilst the MNN in wt parasites is comprised of elongated nanotubules, a dramatic change of vacuolar space architecture was observed in the absence of ASP5. In contrast to RH parasites, the PV of RHΔasp5 parasites did not exhibit a typical MNN which is usually constituted of many long and intricate tubules. Instead, RHΔasp5 parasites contained vesicles and small tubules sparsely distrubuted throughout the vacuolar space (Figs 2B and S4). This indicates that parasites lacking ASP5 are unable to assemble an elaborated MNN. The PV lumen of RHΔasp5 parasites also appears different to that of parasites depleted in both GRA2 and GRA6 [13].

Bottom Line: We show that deletion of ASP5 causes a significant loss in parasite fitness in vitro and an altered virulence in vivo.Additionally, hypermigration of dendritic cells and bradyzoite cyst wall formation are impaired, critically impacting on parasite dissemination and persistence.Overall, the absence of ASP5 dramatically compromises the parasite's ability to modulate host signalling pathways and immune responses.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Molecular Medicine, Centre Médical Universitaire, University of Geneva, Geneva, Switzerland.

ABSTRACT
Toxoplasma gondii possesses sets of dense granule proteins (GRAs) that either assemble at, or cross the parasitophorous vacuole membrane (PVM) and exhibit motifs resembling the HT/PEXEL previously identified in a repertoire of exported Plasmodium proteins. Within Plasmodium spp., cleavage of the HT/PEXEL motif by the endoplasmic reticulum-resident protease Plasmepsin V precedes trafficking to and export across the PVM of proteins involved in pathogenicity and host cell remodelling. Here, we have functionally characterized the T. gondii aspartyl protease 5 (ASP5), a Golgi-resident protease that is phylogenetically related to Plasmepsin V. We show that deletion of ASP5 causes a significant loss in parasite fitness in vitro and an altered virulence in vivo. Furthermore, we reveal that ASP5 is necessary for the cleavage of GRA16, GRA19 and GRA20 at the PEXEL-like motif. In the absence of ASP5, the intravacuolar nanotubular network disappears and several GRAs fail to localize to the PVM, while GRA16 and GRA24, both known to be targeted to the host cell nucleus, are retained within the vacuolar space. Additionally, hypermigration of dendritic cells and bradyzoite cyst wall formation are impaired, critically impacting on parasite dissemination and persistence. Overall, the absence of ASP5 dramatically compromises the parasite's ability to modulate host signalling pathways and immune responses.

No MeSH data available.


Related in: MedlinePlus