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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 affects dendritic cell (DCs) transmigration but not hypermotility.(A) Cell track analyses of DCs were assessed 6–8 hr post-infection for each condition. Circles represent average speed (μm/min) ± s.d. of 100 single cells measured during 45 min from one representative experiment (*P<0.01, Student’s t-test). (B) Transmigration of DCs was assessed in a transwell system +/- CCL19 16 hr post-infection with either type I or type II parasites. CCL19 induction resulted in the transmigration of wt or complemented parasites whereas in Δasp5 strains, transmigration was reduced almost to the same level as the non-induced samples. The graph shows one representative condition ± s.d. from three independent biological replicates (*P<0.01, Student’s t-test).
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ppat.1005211.g007: ASP5 affects dendritic cell (DCs) transmigration but not hypermotility.(A) Cell track analyses of DCs were assessed 6–8 hr post-infection for each condition. Circles represent average speed (μm/min) ± s.d. of 100 single cells measured during 45 min from one representative experiment (*P<0.01, Student’s t-test). (B) Transmigration of DCs was assessed in a transwell system +/- CCL19 16 hr post-infection with either type I or type II parasites. CCL19 induction resulted in the transmigration of wt or complemented parasites whereas in Δasp5 strains, transmigration was reduced almost to the same level as the non-induced samples. The graph shows one representative condition ± s.d. from three independent biological replicates (*P<0.01, Student’s t-test).

Mentions: T. gondii tachyzoites can cross biological barriers [31, 32], however, once inside the host the precise mechanisms leading to systemic dissemination of the parasites remain unknown. T. gondii can exploit the migratory properties of dendritic cells (DC) to spread throughout the organism using the “Trojan horse” strategy. Specifically, upon infection by tachyzoites, DCs exhibit a hypermigratory phenotype [33, 34]. GRA5 has been described as one of the parasite effector molecules capable of increasing the migratory properties of DCs via CCR7 expression without DC activation [35]. To determine the potential impact of ASP5 in this process we first assessed the hypermotility phenotype and observed no significant differences between Δasp5 and the corresponding parental lines either in RH or PRU parasites (Fig 7A). We then conducted a transmigration assay, whereby we measured the ability of infected DCs to migrate in response to CCL19, a CCR7 ligand [34]. In this assay both RHΔasp5 and PRUΔku80Δasp5 parasites showed a considerable reduction in transmigration of infected DCs (Fig 7B).


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 affects dendritic cell (DCs) transmigration but not hypermotility.(A) Cell track analyses of DCs were assessed 6–8 hr post-infection for each condition. Circles represent average speed (μm/min) ± s.d. of 100 single cells measured during 45 min from one representative experiment (*P<0.01, Student’s t-test). (B) Transmigration of DCs was assessed in a transwell system +/- CCL19 16 hr post-infection with either type I or type II parasites. CCL19 induction resulted in the transmigration of wt or complemented parasites whereas in Δasp5 strains, transmigration was reduced almost to the same level as the non-induced samples. The graph shows one representative condition ± s.d. from three independent biological replicates (*P<0.01, Student’s t-test).
© Copyright Policy
Related In: Results  -  Collection

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

ppat.1005211.g007: ASP5 affects dendritic cell (DCs) transmigration but not hypermotility.(A) Cell track analyses of DCs were assessed 6–8 hr post-infection for each condition. Circles represent average speed (μm/min) ± s.d. of 100 single cells measured during 45 min from one representative experiment (*P<0.01, Student’s t-test). (B) Transmigration of DCs was assessed in a transwell system +/- CCL19 16 hr post-infection with either type I or type II parasites. CCL19 induction resulted in the transmigration of wt or complemented parasites whereas in Δasp5 strains, transmigration was reduced almost to the same level as the non-induced samples. The graph shows one representative condition ± s.d. from three independent biological replicates (*P<0.01, Student’s t-test).
Mentions: T. gondii tachyzoites can cross biological barriers [31, 32], however, once inside the host the precise mechanisms leading to systemic dissemination of the parasites remain unknown. T. gondii can exploit the migratory properties of dendritic cells (DC) to spread throughout the organism using the “Trojan horse” strategy. Specifically, upon infection by tachyzoites, DCs exhibit a hypermigratory phenotype [33, 34]. GRA5 has been described as one of the parasite effector molecules capable of increasing the migratory properties of DCs via CCR7 expression without DC activation [35]. To determine the potential impact of ASP5 in this process we first assessed the hypermotility phenotype and observed no significant differences between Δasp5 and the corresponding parental lines either in RH or PRU parasites (Fig 7A). We then conducted a transmigration assay, whereby we measured the ability of infected DCs to migrate in response to CCL19, a CCR7 ligand [34]. In this assay both RHΔasp5 and PRUΔku80Δasp5 parasites showed a considerable reduction in transmigration of infected DCs (Fig 7B).

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