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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

Cyst wall formation is affected in type II parasites lacking ASP5.(A) Induced bradyzoite differentiation of type II parasites under high pH conditions. In parasites lacking ASP5, cyst wall formation, as visualized with DBA lectin, was already affected 1 week after induced-differentiation. α-SAG4 (surface antigen 4) and α-BAG1 (bradyzoite antigen 1) were used as bradyzoite markers. GFP is under the control of a bradyzoite lactate dehydrogenase 2 (LDH2) promoter. Scale bars represent 2 μm. (B) Quantification of the representative IFAs depicted in panel (A). Data are mean value ± s.d. of three independent experiments.
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ppat.1005211.g008: Cyst wall formation is affected in type II parasites lacking ASP5.(A) Induced bradyzoite differentiation of type II parasites under high pH conditions. In parasites lacking ASP5, cyst wall formation, as visualized with DBA lectin, was already affected 1 week after induced-differentiation. α-SAG4 (surface antigen 4) and α-BAG1 (bradyzoite antigen 1) were used as bradyzoite markers. GFP is under the control of a bradyzoite lactate dehydrogenase 2 (LDH2) promoter. Scale bars represent 2 μm. (B) Quantification of the representative IFAs depicted in panel (A). Data are mean value ± s.d. of three independent experiments.

Mentions: Importantly, GRAs are anticipated to play key roles in other stages of the parasite and notably during cyst wall formation, a process that is central for parasite persistence and transmission [36]. To assess the role of ASP5 in cyst wall formation, we used the fluorescent Dolichos biflorus Agglutinin (DBA) lectin to detect the glycosylated protein CST1 (one of the few available markers of the T. gondii cyst wall) [37], following pH induced differentiation of PRUΔku80Δasp5 strain parasites in vitro. Stage conversion from tachyzoites to bradyzoites was measured by expression of SAG4 or BAG1. This conversion took place normally in PRUΔku80Δasp5 parasites and CST1 was also produced, glycosylated and targeted to the PV However, upon closer inspection of the IFA, it became obvious that cyst wall formation was already impaired just one week after induction of differentiation, and even more strikingly impaired two weeks later (Fig 8A and 8B).


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)

Cyst wall formation is affected in type II parasites lacking ASP5.(A) Induced bradyzoite differentiation of type II parasites under high pH conditions. In parasites lacking ASP5, cyst wall formation, as visualized with DBA lectin, was already affected 1 week after induced-differentiation. α-SAG4 (surface antigen 4) and α-BAG1 (bradyzoite antigen 1) were used as bradyzoite markers. GFP is under the control of a bradyzoite lactate dehydrogenase 2 (LDH2) promoter. Scale bars represent 2 μm. (B) Quantification of the representative IFAs depicted in panel (A). Data are mean value ± s.d. of three independent experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4608785&req=5

ppat.1005211.g008: Cyst wall formation is affected in type II parasites lacking ASP5.(A) Induced bradyzoite differentiation of type II parasites under high pH conditions. In parasites lacking ASP5, cyst wall formation, as visualized with DBA lectin, was already affected 1 week after induced-differentiation. α-SAG4 (surface antigen 4) and α-BAG1 (bradyzoite antigen 1) were used as bradyzoite markers. GFP is under the control of a bradyzoite lactate dehydrogenase 2 (LDH2) promoter. Scale bars represent 2 μm. (B) Quantification of the representative IFAs depicted in panel (A). Data are mean value ± s.d. of three independent experiments.
Mentions: Importantly, GRAs are anticipated to play key roles in other stages of the parasite and notably during cyst wall formation, a process that is central for parasite persistence and transmission [36]. To assess the role of ASP5 in cyst wall formation, we used the fluorescent Dolichos biflorus Agglutinin (DBA) lectin to detect the glycosylated protein CST1 (one of the few available markers of the T. gondii cyst wall) [37], following pH induced differentiation of PRUΔku80Δasp5 strain parasites in vitro. Stage conversion from tachyzoites to bradyzoites was measured by expression of SAG4 or BAG1. This conversion took place normally in PRUΔku80Δasp5 parasites and CST1 was also produced, glycosylated and targeted to the PV However, upon closer inspection of the IFA, it became obvious that cyst wall formation was already impaired just one week after induction of differentiation, and even more strikingly impaired two weeks later (Fig 8A and 8B).

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