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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

ASP5 is a dispensable Golgi-resident protein.(A) In type I parasites, indirect immunofluorescence analyses (IFA) using α-Ty antibodies revealed that ASP5-3Ty is a Golgi-resident protein, which co-localizes with the Golgi marker GRASP-YFP. Gliding associated protein 45 (α-GAP45) antibodies were used to stain the parasite periphery. Scale bars represent 2 μm. (B) Western blots completed with α-Ty antibodies, revealed that ASP5-3Ty migrates at the molecular size of 100 kDa, but also as a second lower band of 55 kDa. No Ty signal was detected in RHΔku80Δasp5 parasites. Complementation with ASP5-Ty gDNA (asp5g-Ty) restores the wt pattern while complementation with ASP5-Ty cDNA (asp5c-Ty) leads only to production of the 100 kDa band. T. gondii Actin 1 (α-ACT1) was used as a loading control. (C) In type II parasites, ASP5-3Ty migrates similarly to that seen in type I parasites, with two bands detected at 100 and 55 kDa. No Ty signal was detected for PRUΔku80Δasp5 parasites. Detection of α-ACT1 was used as a loading control. (D) Intracellular growth type I and type II parasites was assessed after 24 hr in complete media. Parasites lacking ASP5 were not impacted in their ability to replicate intracellularly. Data are mean value ± s.d. of three independent experiments. (E) Deletion of ASP5 resulted in a significant impairment of the lytic cycle, as assessed by plaque formation after 7 days, in both type I and II parasites. Complementation with ASP5-Ty gDNA (asp5g-Ty) fully restored plaque formation whereas complementation with ASP5-Ty cDNA (asp5c-Ty) led to an intermediate plaque phenotype. (F) Mean area of 10 plaques ± s.d.is depicted. (G) Quantification of BIPPO-stimulated egressed vacuoles. Data show mean ± s.d. of three independent experiments. (H) During natural egress, a significant fraction of the parasites remains trapped either inside the parasitophorous vacuole or the host plasma membrane which resemble detached sphere-like structures. No other apparent phenotype was observed.
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ppat.1005211.g001: ASP5 is a dispensable Golgi-resident protein.(A) In type I parasites, indirect immunofluorescence analyses (IFA) using α-Ty antibodies revealed that ASP5-3Ty is a Golgi-resident protein, which co-localizes with the Golgi marker GRASP-YFP. Gliding associated protein 45 (α-GAP45) antibodies were used to stain the parasite periphery. Scale bars represent 2 μm. (B) Western blots completed with α-Ty antibodies, revealed that ASP5-3Ty migrates at the molecular size of 100 kDa, but also as a second lower band of 55 kDa. No Ty signal was detected in RHΔku80Δasp5 parasites. Complementation with ASP5-Ty gDNA (asp5g-Ty) restores the wt pattern while complementation with ASP5-Ty cDNA (asp5c-Ty) leads only to production of the 100 kDa band. T. gondii Actin 1 (α-ACT1) was used as a loading control. (C) In type II parasites, ASP5-3Ty migrates similarly to that seen in type I parasites, with two bands detected at 100 and 55 kDa. No Ty signal was detected for PRUΔku80Δasp5 parasites. Detection of α-ACT1 was used as a loading control. (D) Intracellular growth type I and type II parasites was assessed after 24 hr in complete media. Parasites lacking ASP5 were not impacted in their ability to replicate intracellularly. Data are mean value ± s.d. of three independent experiments. (E) Deletion of ASP5 resulted in a significant impairment of the lytic cycle, as assessed by plaque formation after 7 days, in both type I and II parasites. Complementation with ASP5-Ty gDNA (asp5g-Ty) fully restored plaque formation whereas complementation with ASP5-Ty cDNA (asp5c-Ty) led to an intermediate plaque phenotype. (F) Mean area of 10 plaques ± s.d.is depicted. (G) Quantification of BIPPO-stimulated egressed vacuoles. Data show mean ± s.d. of three independent experiments. (H) During natural egress, a significant fraction of the parasites remains trapped either inside the parasitophorous vacuole or the host plasma membrane which resemble detached sphere-like structures. No other apparent phenotype was observed.

Mentions: ASP5 has previously been described as a Golgi-resident protein when expressed as an epitope-tagged second copy [19]. To determine its role and importance in T. gondii, we first confirmed the localization of ASP5 by inserting a 3Ty-epitope tag at the carboxyl-terminus of the endogenous ASP5 locus in both type I (RHΔku80) and type II (Prugniaud, PRUΔku80) strains. Endogenous ASP5-3Ty co-localizes with the Golgi marker GRASP (Fig 1A) and shows two forms by western blot analyses that have not been previously reported. The 100 kDa band is in agreement with the predicted full length protein size (108 kDa), whereas a smaller form migrates with an apparent molecular weight of 55 kDa (short-ASP5). 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 35S-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).


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)

ASP5 is a dispensable Golgi-resident protein.(A) In type I parasites, indirect immunofluorescence analyses (IFA) using α-Ty antibodies revealed that ASP5-3Ty is a Golgi-resident protein, which co-localizes with the Golgi marker GRASP-YFP. Gliding associated protein 45 (α-GAP45) antibodies were used to stain the parasite periphery. Scale bars represent 2 μm. (B) Western blots completed with α-Ty antibodies, revealed that ASP5-3Ty migrates at the molecular size of 100 kDa, but also as a second lower band of 55 kDa. No Ty signal was detected in RHΔku80Δasp5 parasites. Complementation with ASP5-Ty gDNA (asp5g-Ty) restores the wt pattern while complementation with ASP5-Ty cDNA (asp5c-Ty) leads only to production of the 100 kDa band. T. gondii Actin 1 (α-ACT1) was used as a loading control. (C) In type II parasites, ASP5-3Ty migrates similarly to that seen in type I parasites, with two bands detected at 100 and 55 kDa. No Ty signal was detected for PRUΔku80Δasp5 parasites. Detection of α-ACT1 was used as a loading control. (D) Intracellular growth type I and type II parasites was assessed after 24 hr in complete media. Parasites lacking ASP5 were not impacted in their ability to replicate intracellularly. Data are mean value ± s.d. of three independent experiments. (E) Deletion of ASP5 resulted in a significant impairment of the lytic cycle, as assessed by plaque formation after 7 days, in both type I and II parasites. Complementation with ASP5-Ty gDNA (asp5g-Ty) fully restored plaque formation whereas complementation with ASP5-Ty cDNA (asp5c-Ty) led to an intermediate plaque phenotype. (F) Mean area of 10 plaques ± s.d.is depicted. (G) Quantification of BIPPO-stimulated egressed vacuoles. Data show mean ± s.d. of three independent experiments. (H) During natural egress, a significant fraction of the parasites remains trapped either inside the parasitophorous vacuole or the host plasma membrane which resemble detached sphere-like structures. No other apparent phenotype was observed.
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Related In: Results  -  Collection

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

ppat.1005211.g001: ASP5 is a dispensable Golgi-resident protein.(A) In type I parasites, indirect immunofluorescence analyses (IFA) using α-Ty antibodies revealed that ASP5-3Ty is a Golgi-resident protein, which co-localizes with the Golgi marker GRASP-YFP. Gliding associated protein 45 (α-GAP45) antibodies were used to stain the parasite periphery. Scale bars represent 2 μm. (B) Western blots completed with α-Ty antibodies, revealed that ASP5-3Ty migrates at the molecular size of 100 kDa, but also as a second lower band of 55 kDa. No Ty signal was detected in RHΔku80Δasp5 parasites. Complementation with ASP5-Ty gDNA (asp5g-Ty) restores the wt pattern while complementation with ASP5-Ty cDNA (asp5c-Ty) leads only to production of the 100 kDa band. T. gondii Actin 1 (α-ACT1) was used as a loading control. (C) In type II parasites, ASP5-3Ty migrates similarly to that seen in type I parasites, with two bands detected at 100 and 55 kDa. No Ty signal was detected for PRUΔku80Δasp5 parasites. Detection of α-ACT1 was used as a loading control. (D) Intracellular growth type I and type II parasites was assessed after 24 hr in complete media. Parasites lacking ASP5 were not impacted in their ability to replicate intracellularly. Data are mean value ± s.d. of three independent experiments. (E) Deletion of ASP5 resulted in a significant impairment of the lytic cycle, as assessed by plaque formation after 7 days, in both type I and II parasites. Complementation with ASP5-Ty gDNA (asp5g-Ty) fully restored plaque formation whereas complementation with ASP5-Ty cDNA (asp5c-Ty) led to an intermediate plaque phenotype. (F) Mean area of 10 plaques ± s.d.is depicted. (G) Quantification of BIPPO-stimulated egressed vacuoles. Data show mean ± s.d. of three independent experiments. (H) During natural egress, a significant fraction of the parasites remains trapped either inside the parasitophorous vacuole or the host plasma membrane which resemble detached sphere-like structures. No other apparent phenotype was observed.
Mentions: ASP5 has previously been described as a Golgi-resident protein when expressed as an epitope-tagged second copy [19]. To determine its role and importance in T. gondii, we first confirmed the localization of ASP5 by inserting a 3Ty-epitope tag at the carboxyl-terminus of the endogenous ASP5 locus in both type I (RHΔku80) and type II (Prugniaud, PRUΔku80) strains. Endogenous ASP5-3Ty co-localizes with the Golgi marker GRASP (Fig 1A) and shows two forms by western blot analyses that have not been previously reported. The 100 kDa band is in agreement with the predicted full length protein size (108 kDa), whereas a smaller form migrates with an apparent molecular weight of 55 kDa (short-ASP5). 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 35S-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).

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