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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 deletion compromises the export of GRA16 and GRA24 beyond the PVM.(A) In the absence of ASP5, GRA16-3Myc transiently transfected in type I parasites was no longer addressed to the host nucleus (white arrowheads) and instead remained within the PV. Scale bars represent 2 μm. (B) The PEXEL-like motif-containing GRA16-3Myc displays an altered cleavage profile in RHΔasp5 and RHΔasp5/asp5c-D/A parasites as detected by western blot analyses. R1 and R2: repeated region. (C) IFAs of a stably expressed second copy of GRA24-Myc revealed that, in parasites lacking ASP5, this protein was no longer addressed to the host nucleus and instead accumulates within the PV. (D) GRA24-Myc, which is devoid of PEXEL-like sequence, shows a similar migration profile in both strains. For panel B and D, parasites were collected 24 hr post-infection. α-Catalase was used as a loading control.
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ppat.1005211.g004: ASP5 deletion compromises the export of GRA16 and GRA24 beyond the PVM.(A) In the absence of ASP5, GRA16-3Myc transiently transfected in type I parasites was no longer addressed to the host nucleus (white arrowheads) and instead remained within the PV. Scale bars represent 2 μm. (B) The PEXEL-like motif-containing GRA16-3Myc displays an altered cleavage profile in RHΔasp5 and RHΔasp5/asp5c-D/A parasites as detected by western blot analyses. R1 and R2: repeated region. (C) IFAs of a stably expressed second copy of GRA24-Myc revealed that, in parasites lacking ASP5, this protein was no longer addressed to the host nucleus and instead accumulates within the PV. (D) GRA24-Myc, which is devoid of PEXEL-like sequence, shows a similar migration profile in both strains. For panel B and D, parasites were collected 24 hr post-infection. α-Catalase was used as a loading control.

Mentions: Following parasite internalization and the concomitant PVM formation, two GRAs are known to cross the PVM and be exported into the host cell nucleus [16, 17]. To assess the fate of GRA16 and GRA24, second copies of GRA24 driven by a tubulin promoter and GRA16 driven by its endogenous promoter and fused to 3 Myc tags were expressed in type I parasites (Fig 4A and 4C). As previously reported, GRA16 and GRA24 show a dual localization in the PV as well as the host cell nucleus in RH parasites. In sharp contrast, in RHΔasp5 parasites both GRAs accumulate in the PV but fail to reach the host nucleus, even at a high MOI. Whereas ASP5 cDNA or gDNA complementation restored the host cell nuclear localization, the catalytically inactive ASP5D/A is not sufficient to promote GRA16 export (Fig 4A). In wild type parasites GRA16-3Myc showed two forms that presumably correspond to unprocessed and processed forms given the fact that the protein possesses a PEXEL-like motif starting at the arginine residue in position 63, corresponding after cleavage to a drop of ~5 kDa. Contrastingly, in RHΔasp5 parasites the unprocessed form of GRA16 (which migrated slightly slower) strongly accumulates while a residual level of the processed form was still detectable (Fig 4B). This might result from the action of a different protease, or may represent a degradation product. The small shift in the unprocessed band observed between RH and RHΔasp5 parasites cannot be explained and will require further investigation.


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 deletion compromises the export of GRA16 and GRA24 beyond the PVM.(A) In the absence of ASP5, GRA16-3Myc transiently transfected in type I parasites was no longer addressed to the host nucleus (white arrowheads) and instead remained within the PV. Scale bars represent 2 μm. (B) The PEXEL-like motif-containing GRA16-3Myc displays an altered cleavage profile in RHΔasp5 and RHΔasp5/asp5c-D/A parasites as detected by western blot analyses. R1 and R2: repeated region. (C) IFAs of a stably expressed second copy of GRA24-Myc revealed that, in parasites lacking ASP5, this protein was no longer addressed to the host nucleus and instead accumulates within the PV. (D) GRA24-Myc, which is devoid of PEXEL-like sequence, shows a similar migration profile in both strains. For panel B and D, parasites were collected 24 hr post-infection. α-Catalase was used as a loading control.
© Copyright Policy
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC4608785&req=5

ppat.1005211.g004: ASP5 deletion compromises the export of GRA16 and GRA24 beyond the PVM.(A) In the absence of ASP5, GRA16-3Myc transiently transfected in type I parasites was no longer addressed to the host nucleus (white arrowheads) and instead remained within the PV. Scale bars represent 2 μm. (B) The PEXEL-like motif-containing GRA16-3Myc displays an altered cleavage profile in RHΔasp5 and RHΔasp5/asp5c-D/A parasites as detected by western blot analyses. R1 and R2: repeated region. (C) IFAs of a stably expressed second copy of GRA24-Myc revealed that, in parasites lacking ASP5, this protein was no longer addressed to the host nucleus and instead accumulates within the PV. (D) GRA24-Myc, which is devoid of PEXEL-like sequence, shows a similar migration profile in both strains. For panel B and D, parasites were collected 24 hr post-infection. α-Catalase was used as a loading control.
Mentions: Following parasite internalization and the concomitant PVM formation, two GRAs are known to cross the PVM and be exported into the host cell nucleus [16, 17]. To assess the fate of GRA16 and GRA24, second copies of GRA24 driven by a tubulin promoter and GRA16 driven by its endogenous promoter and fused to 3 Myc tags were expressed in type I parasites (Fig 4A and 4C). As previously reported, GRA16 and GRA24 show a dual localization in the PV as well as the host cell nucleus in RH parasites. In sharp contrast, in RHΔasp5 parasites both GRAs accumulate in the PV but fail to reach the host nucleus, even at a high MOI. Whereas ASP5 cDNA or gDNA complementation restored the host cell nuclear localization, the catalytically inactive ASP5D/A is not sufficient to promote GRA16 export (Fig 4A). In wild type parasites GRA16-3Myc showed two forms that presumably correspond to unprocessed and processed forms given the fact that the protein possesses a PEXEL-like motif starting at the arginine residue in position 63, corresponding after cleavage to a drop of ~5 kDa. Contrastingly, in RHΔasp5 parasites the unprocessed form of GRA16 (which migrated slightly slower) strongly accumulates while a residual level of the processed form was still detectable (Fig 4B). This might result from the action of a different protease, or may represent a degradation product. The small shift in the unprocessed band observed between RH and RHΔasp5 parasites cannot be explained and will require further investigation.

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