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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

Stage specific functions of ASP5 in T. gondii.(A) Schematic representation of a host cell infected by either type I or type II tachyzoites. In a wt situation (left panel), intracellular tachyzoites reside within an MNN containing PV. During intracellular development, dense granule proteins are delivered either to the vacuolar space, the PVM, or are addressed to the host cell cytosol and nucleus. Intriguingly, the absence of ASP5 (right panel) does not affect the secretion of the dense granules, and accordingly, PEXEL-like and non-PEXEL-like motif-containing proteins are still delivered to the PV. However, ASP5 deletion leads to the disappearance of the MNN, altered localization of some PV- and PVM-resident GRAs, and to the PV-accumulation of proteins normally exported to the host cell cytosol. Accordingly, the overall infected host cell transcription profile response is significantly different in Δasp5 parasites infection. The accumulation of some exported GRAs in the PV suggests that the export machinery used to cross the PVM is affected upon ASP5 deletion. In contrast, no significant effect was observed for early host organelle recruitment to the PVM, but only a reduced fusion of host mitochondria was observed. Specifically in type I parasites, blocking of IRG recruitment is unaffected. SAG1 is a tachyzoite marker. (B) Differentiation in bradyzoites is not affected by ASP5 deletion but parasites fail to form a cyst wall. SAG4 and BAG1 are bradyzoite markers.
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ppat.1005211.g009: Stage specific functions of ASP5 in T. gondii.(A) Schematic representation of a host cell infected by either type I or type II tachyzoites. In a wt situation (left panel), intracellular tachyzoites reside within an MNN containing PV. During intracellular development, dense granule proteins are delivered either to the vacuolar space, the PVM, or are addressed to the host cell cytosol and nucleus. Intriguingly, the absence of ASP5 (right panel) does not affect the secretion of the dense granules, and accordingly, PEXEL-like and non-PEXEL-like motif-containing proteins are still delivered to the PV. However, ASP5 deletion leads to the disappearance of the MNN, altered localization of some PV- and PVM-resident GRAs, and to the PV-accumulation of proteins normally exported to the host cell cytosol. Accordingly, the overall infected host cell transcription profile response is significantly different in Δasp5 parasites infection. The accumulation of some exported GRAs in the PV suggests that the export machinery used to cross the PVM is affected upon ASP5 deletion. In contrast, no significant effect was observed for early host organelle recruitment to the PVM, but only a reduced fusion of host mitochondria was observed. Specifically in type I parasites, blocking of IRG recruitment is unaffected. SAG1 is a tachyzoite marker. (B) Differentiation in bradyzoites is not affected by ASP5 deletion but parasites fail to form a cyst wall. SAG4 and BAG1 are bradyzoite markers.

Mentions: Here we report the characterisation of the Golgi-resident protease ASP5, which is responsible for the cleavage of PEXEL-like motif-containing proteins in T. gondii. Whilst Plasmepsin V appears to be primarily dedicated to cleavage of proteins destined to be exported beyond the PVM, deletion of ASP5 causes considerable pleiotropic effects by effecting both exported and PV/PVM-resident proteins (Fig 9). Accordingly and in contrast to P. falciparum, numerous PEXEL-like containing proteins remain within the PV and are not further exported. Deletion of ASP5, without affecting dense granule secretion, caused significant morphological aberrations of the PV, most notably being the defect in MNN formation. The role of this elaborated structure is still mysterious, although it is presumed to participate in parasite access to host cell nutrients. In this context, and rather unexpectedly, depletion of ASP5 does not appear to impose any restriction on intracellular parasite replication even in glucose depleted media. The molecular connection between ASP5 activity and MNN formation is not known, however such a phenotype was previously described when individual GRAs were knocked out [13]. Alternatively, the MNN could participate in the process of egress, which is unexpectedly affected in parasites lacking ASP5. It is known that some GRAs form high molecular weight complexes within the dense granules [44] and also exist as heteromeric complexes in the PV [44]. It is therefore conceivable that deletion of ASP5 could affect formation of these complexes and thus ASP5 will not only affect the activity of its direct substrates but also their interacting partners.


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)

Stage specific functions of ASP5 in T. gondii.(A) Schematic representation of a host cell infected by either type I or type II tachyzoites. In a wt situation (left panel), intracellular tachyzoites reside within an MNN containing PV. During intracellular development, dense granule proteins are delivered either to the vacuolar space, the PVM, or are addressed to the host cell cytosol and nucleus. Intriguingly, the absence of ASP5 (right panel) does not affect the secretion of the dense granules, and accordingly, PEXEL-like and non-PEXEL-like motif-containing proteins are still delivered to the PV. However, ASP5 deletion leads to the disappearance of the MNN, altered localization of some PV- and PVM-resident GRAs, and to the PV-accumulation of proteins normally exported to the host cell cytosol. Accordingly, the overall infected host cell transcription profile response is significantly different in Δasp5 parasites infection. The accumulation of some exported GRAs in the PV suggests that the export machinery used to cross the PVM is affected upon ASP5 deletion. In contrast, no significant effect was observed for early host organelle recruitment to the PVM, but only a reduced fusion of host mitochondria was observed. Specifically in type I parasites, blocking of IRG recruitment is unaffected. SAG1 is a tachyzoite marker. (B) Differentiation in bradyzoites is not affected by ASP5 deletion but parasites fail to form a cyst wall. SAG4 and BAG1 are bradyzoite markers.
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Related In: Results  -  Collection

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ppat.1005211.g009: Stage specific functions of ASP5 in T. gondii.(A) Schematic representation of a host cell infected by either type I or type II tachyzoites. In a wt situation (left panel), intracellular tachyzoites reside within an MNN containing PV. During intracellular development, dense granule proteins are delivered either to the vacuolar space, the PVM, or are addressed to the host cell cytosol and nucleus. Intriguingly, the absence of ASP5 (right panel) does not affect the secretion of the dense granules, and accordingly, PEXEL-like and non-PEXEL-like motif-containing proteins are still delivered to the PV. However, ASP5 deletion leads to the disappearance of the MNN, altered localization of some PV- and PVM-resident GRAs, and to the PV-accumulation of proteins normally exported to the host cell cytosol. Accordingly, the overall infected host cell transcription profile response is significantly different in Δasp5 parasites infection. The accumulation of some exported GRAs in the PV suggests that the export machinery used to cross the PVM is affected upon ASP5 deletion. In contrast, no significant effect was observed for early host organelle recruitment to the PVM, but only a reduced fusion of host mitochondria was observed. Specifically in type I parasites, blocking of IRG recruitment is unaffected. SAG1 is a tachyzoite marker. (B) Differentiation in bradyzoites is not affected by ASP5 deletion but parasites fail to form a cyst wall. SAG4 and BAG1 are bradyzoite markers.
Mentions: Here we report the characterisation of the Golgi-resident protease ASP5, which is responsible for the cleavage of PEXEL-like motif-containing proteins in T. gondii. Whilst Plasmepsin V appears to be primarily dedicated to cleavage of proteins destined to be exported beyond the PVM, deletion of ASP5 causes considerable pleiotropic effects by effecting both exported and PV/PVM-resident proteins (Fig 9). Accordingly and in contrast to P. falciparum, numerous PEXEL-like containing proteins remain within the PV and are not further exported. Deletion of ASP5, without affecting dense granule secretion, caused significant morphological aberrations of the PV, most notably being the defect in MNN formation. The role of this elaborated structure is still mysterious, although it is presumed to participate in parasite access to host cell nutrients. In this context, and rather unexpectedly, depletion of ASP5 does not appear to impose any restriction on intracellular parasite replication even in glucose depleted media. The molecular connection between ASP5 activity and MNN formation is not known, however such a phenotype was previously described when individual GRAs were knocked out [13]. Alternatively, the MNN could participate in the process of egress, which is unexpectedly affected in parasites lacking ASP5. It is known that some GRAs form high molecular weight complexes within the dense granules [44] and also exist as heteromeric complexes in the PV [44]. It is therefore conceivable that deletion of ASP5 could affect formation of these complexes and thus ASP5 will not only affect the activity of its direct substrates but also their interacting partners.

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