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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 mediates the cleavage of PEXEL-like motif-containing proteins but does not impact on dense granule secretion.(A) Type I parasites were transiently transfected with GRA19-HA, GRA19-HA-R124A (PEXEL-like mutant control) or GRA20-HA. Intracellular parasites were collected and analysed by western blot. In the absence of ASP5, processing of GRA19-HA and GRA20-HA was abolished as detected by α-HA antibodies. Middle gel represents a longer exposure. α-Catalase antibodies were used as a loading control. (B) IFAs of parasites transiently transfected with GRA19-HA and GRA20-HA as presented in (A). The absence of the ASP5-dependent processing of GRA20 did not affect its localization at the PVM. Scale bars represent 2 μm. (C) Dense granule secretion assays in type I parasites revealed that deletion of ASP5 does not impair GRA1, 3 or 7 secretion. Interestingly, the multiple band pattern of GRA7 is abolished under these conditions. Catalase and MIC2 (micronemal protein 2) were used as controls for the non-secreted and secreted fraction respectively (MIC2 is processed upon secretion of the micronemes). ESA: excretory secretory antigens.
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ppat.1005211.g003: ASP5 mediates the cleavage of PEXEL-like motif-containing proteins but does not impact on dense granule secretion.(A) Type I parasites were transiently transfected with GRA19-HA, GRA19-HA-R124A (PEXEL-like mutant control) or GRA20-HA. Intracellular parasites were collected and analysed by western blot. In the absence of ASP5, processing of GRA19-HA and GRA20-HA was abolished as detected by α-HA antibodies. Middle gel represents a longer exposure. α-Catalase antibodies were used as a loading control. (B) IFAs of parasites transiently transfected with GRA19-HA and GRA20-HA as presented in (A). The absence of the ASP5-dependent processing of GRA20 did not affect its localization at the PVM. Scale bars represent 2 μm. (C) Dense granule secretion assays in type I parasites revealed that deletion of ASP5 does not impair GRA1, 3 or 7 secretion. Interestingly, the multiple band pattern of GRA7 is abolished under these conditions. Catalase and MIC2 (micronemal protein 2) were used as controls for the non-secreted and secreted fraction respectively (MIC2 is processed upon secretion of the micronemes). ESA: excretory secretory antigens.

Mentions: The recently described GRA19 and GRA20 were investigated here via expression of C-terminally HA-tagged second copies as previously described [18]. Both proteins are known to be processed within their PEXEL-like motif by an unidentified protease [18]. Transiently expressed GRA19-HA and GRA20-HA were modestly processed as previously observed in parental parasites, however this cleavage was abolished in the absence of ASP5 (Fig 3A). An R/A point mutant in the GRA19 PEXEL motif prevented processing as previously reported, and served here as a control. The absence of ASP5 did not alter the localization of either GRA19 or GRA20 in an obvious manner as documented by IFA (Fig 3B). Given that both processing and localization of several GRAs is affected by the absence of ASP5, we examined whether the overall secretion by dense granules was impaired. We developed a secretion assay whereby released GRAs were collected from the supernatant of extracellular parasites and referred to here as excretory secretory antigens (ESA) upon western blot analyses. Secretion of the microneme protein MIC2 was used here as a control for parasite viability and fitness. These assays revealed that secretion of GRA1, 2, 3 and 7 were comparable in RH and RHΔasp5 strain parasites (Fig 3C). Interestingly, GRA7 which was previously reported to be phosphorylated by an unidentified host cell kinase [22, 23] gave rise to a ladder of bands which appears to be extensively reduced or even abolished in the absence of ASP5 (Fig 3C). This suggests that GRA7 may be subtly miss-targeted in the absence of ASP5 and hence no longer accessible to the host kinase. Taken together, these results indicate that ASP5 is responsible for the cleavage of some PVM-enclosed GRAs and in its absence, these proteins are normally secreted by the dense granules yet are impacted in their final destination. This is likely to lead to defects in post-translational modifications (e.g. phosphorylation of GRA7) and altered protein activity given that the MNN is no longer formed.


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 mediates the cleavage of PEXEL-like motif-containing proteins but does not impact on dense granule secretion.(A) Type I parasites were transiently transfected with GRA19-HA, GRA19-HA-R124A (PEXEL-like mutant control) or GRA20-HA. Intracellular parasites were collected and analysed by western blot. In the absence of ASP5, processing of GRA19-HA and GRA20-HA was abolished as detected by α-HA antibodies. Middle gel represents a longer exposure. α-Catalase antibodies were used as a loading control. (B) IFAs of parasites transiently transfected with GRA19-HA and GRA20-HA as presented in (A). The absence of the ASP5-dependent processing of GRA20 did not affect its localization at the PVM. Scale bars represent 2 μm. (C) Dense granule secretion assays in type I parasites revealed that deletion of ASP5 does not impair GRA1, 3 or 7 secretion. Interestingly, the multiple band pattern of GRA7 is abolished under these conditions. Catalase and MIC2 (micronemal protein 2) were used as controls for the non-secreted and secreted fraction respectively (MIC2 is processed upon secretion of the micronemes). ESA: excretory secretory antigens.
© Copyright Policy
Related In: Results  -  Collection

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

ppat.1005211.g003: ASP5 mediates the cleavage of PEXEL-like motif-containing proteins but does not impact on dense granule secretion.(A) Type I parasites were transiently transfected with GRA19-HA, GRA19-HA-R124A (PEXEL-like mutant control) or GRA20-HA. Intracellular parasites were collected and analysed by western blot. In the absence of ASP5, processing of GRA19-HA and GRA20-HA was abolished as detected by α-HA antibodies. Middle gel represents a longer exposure. α-Catalase antibodies were used as a loading control. (B) IFAs of parasites transiently transfected with GRA19-HA and GRA20-HA as presented in (A). The absence of the ASP5-dependent processing of GRA20 did not affect its localization at the PVM. Scale bars represent 2 μm. (C) Dense granule secretion assays in type I parasites revealed that deletion of ASP5 does not impair GRA1, 3 or 7 secretion. Interestingly, the multiple band pattern of GRA7 is abolished under these conditions. Catalase and MIC2 (micronemal protein 2) were used as controls for the non-secreted and secreted fraction respectively (MIC2 is processed upon secretion of the micronemes). ESA: excretory secretory antigens.
Mentions: The recently described GRA19 and GRA20 were investigated here via expression of C-terminally HA-tagged second copies as previously described [18]. Both proteins are known to be processed within their PEXEL-like motif by an unidentified protease [18]. Transiently expressed GRA19-HA and GRA20-HA were modestly processed as previously observed in parental parasites, however this cleavage was abolished in the absence of ASP5 (Fig 3A). An R/A point mutant in the GRA19 PEXEL motif prevented processing as previously reported, and served here as a control. The absence of ASP5 did not alter the localization of either GRA19 or GRA20 in an obvious manner as documented by IFA (Fig 3B). Given that both processing and localization of several GRAs is affected by the absence of ASP5, we examined whether the overall secretion by dense granules was impaired. We developed a secretion assay whereby released GRAs were collected from the supernatant of extracellular parasites and referred to here as excretory secretory antigens (ESA) upon western blot analyses. Secretion of the microneme protein MIC2 was used here as a control for parasite viability and fitness. These assays revealed that secretion of GRA1, 2, 3 and 7 were comparable in RH and RHΔasp5 strain parasites (Fig 3C). Interestingly, GRA7 which was previously reported to be phosphorylated by an unidentified host cell kinase [22, 23] gave rise to a ladder of bands which appears to be extensively reduced or even abolished in the absence of ASP5 (Fig 3C). This suggests that GRA7 may be subtly miss-targeted in the absence of ASP5 and hence no longer accessible to the host kinase. Taken together, these results indicate that ASP5 is responsible for the cleavage of some PVM-enclosed GRAs and in its absence, these proteins are normally secreted by the dense granules yet are impacted in their final destination. This is likely to lead to defects in post-translational modifications (e.g. phosphorylation of GRA7) and altered protein activity given that the MNN is no longer formed.

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