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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 modulates host innate immune responses.(A) Interferon-γ stimulated, bone marrow derived macrophages (BMDMs) were infected with type I and type II parasite lines for 1 hr at a MOI of 1. In the absence of ASP5, type I parasites were not affected in IRG recruitment to the PVM. Type II parasites were used as controls. Parasites were labelled with α-GRA2, host IRGs with α-Irgb6 antibodies, and host nuclei with DAPI. IFAs are representative data from three independent biological experiments. Scale bars represent 2 μm. (B) IL-12p40 levels were measured by ELISA from supernatant collected 40 hr after BMDM infection with type I and type II strain parasites. Supernatant from uninfected cells was used as a control (u.i.). In both types, the absence of ASP5 dramatically reduces IL-12p40 levels. Data are mean value ± s.d. of three independent experiments. (*P<0.01, **P<0.005, Student’s t-test). (C) Quantitative chemokine production was determined by qRT-PCR on mouse embryonic fibroblast (pMEFs) infected with type I and type II parasites. Values were normalized to the amount of actin in each sample. Data are mean value ± s.d. of three independent experiments. (*P<0.01, **P<0.05, Student’s t-test). (D) Following KEGG analysis of the differentially expressed genes (>2 fold, p-value < 0.05) between parental and Δasp5 infected BMDMs, heatmaps were generated with genes selected from representative KEGG pathways. (Red: low, Green: high). Both up- and down-regulated genes of the host cell were analyzed together.
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ppat.1005211.g006: ASP5 modulates host innate immune responses.(A) Interferon-γ stimulated, bone marrow derived macrophages (BMDMs) were infected with type I and type II parasite lines for 1 hr at a MOI of 1. In the absence of ASP5, type I parasites were not affected in IRG recruitment to the PVM. Type II parasites were used as controls. Parasites were labelled with α-GRA2, host IRGs with α-Irgb6 antibodies, and host nuclei with DAPI. IFAs are representative data from three independent biological experiments. Scale bars represent 2 μm. (B) IL-12p40 levels were measured by ELISA from supernatant collected 40 hr after BMDM infection with type I and type II strain parasites. Supernatant from uninfected cells was used as a control (u.i.). In both types, the absence of ASP5 dramatically reduces IL-12p40 levels. Data are mean value ± s.d. of three independent experiments. (*P<0.01, **P<0.005, Student’s t-test). (C) Quantitative chemokine production was determined by qRT-PCR on mouse embryonic fibroblast (pMEFs) infected with type I and type II parasites. Values were normalized to the amount of actin in each sample. Data are mean value ± s.d. of three independent experiments. (*P<0.01, **P<0.05, Student’s t-test). (D) Following KEGG analysis of the differentially expressed genes (>2 fold, p-value < 0.05) between parental and Δasp5 infected BMDMs, heatmaps were generated with genes selected from representative KEGG pathways. (Red: low, Green: high). Both up- and down-regulated genes of the host cell were analyzed together.

Mentions: One of the key events in establishing a protective Th1 immune response against T. gondii is the ability of host immune cells to produce the pro-inflammatory cytokine interleukin 12 (IL-12), which in turn stimulates the production of interferon gamma (IFNγ) by natural killer (NK) cells, CD4+ and CD8+ T cells [15, 24]. IFNγ is the major pro-inflammatory cytokine driving multiple cellular defense mechanisms during both the acute and chronic phases of infection [25]. Importantly, the immunity related GTPases (IRG proteins) constitute a large family of interferon-inducible proteins that mediate early resistance to T. gondii infection in mice. Several studies have shown that IRGs, in particular Irga6 and Irgb6 are recruited to the nascent PVM, where they cause disruption of the vacuole and parasite death. While the ROP18 complex of type I parasites is able to phosphorylate IRG proteins, thereby preventing their oligomerization and loading onto the PVM, type II parasites are unable to block the action of IRG proteins due to the polymorphic nature of ROP5, which forms part of the ROP18 complex [26]. Recent studies have associated GRA7 to the ROP18 complex by acting as regulator for ROP18-specific inactivation of Irga6 [23, 27]. To determine whether vacuoles containing RHΔasp5 parasites failed to block IRG recruitment to the PVM, an IRG recruitment assay was performed for Irgb6. Our results indicate that RHΔasp5 parasites behave like RH parasites and remain non-susceptible to Irgb6 and Irga6 loading (Fig 6A, left panel). Conversely, Irgb6 was recruited to the PVM of PRUΔku80Δasp5 parasites as previously reported for PRUΔku80 (Fig 6A, right panel) [26]. Given these data and in spite of the impact of ASP5 on GRA7 phosphorylation (which forms part of the ROP18 complex), we propose that ASP5 activity is not essential for the activity of the ROP18 complex [23].


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 modulates host innate immune responses.(A) Interferon-γ stimulated, bone marrow derived macrophages (BMDMs) were infected with type I and type II parasite lines for 1 hr at a MOI of 1. In the absence of ASP5, type I parasites were not affected in IRG recruitment to the PVM. Type II parasites were used as controls. Parasites were labelled with α-GRA2, host IRGs with α-Irgb6 antibodies, and host nuclei with DAPI. IFAs are representative data from three independent biological experiments. Scale bars represent 2 μm. (B) IL-12p40 levels were measured by ELISA from supernatant collected 40 hr after BMDM infection with type I and type II strain parasites. Supernatant from uninfected cells was used as a control (u.i.). In both types, the absence of ASP5 dramatically reduces IL-12p40 levels. Data are mean value ± s.d. of three independent experiments. (*P<0.01, **P<0.005, Student’s t-test). (C) Quantitative chemokine production was determined by qRT-PCR on mouse embryonic fibroblast (pMEFs) infected with type I and type II parasites. Values were normalized to the amount of actin in each sample. Data are mean value ± s.d. of three independent experiments. (*P<0.01, **P<0.05, Student’s t-test). (D) Following KEGG analysis of the differentially expressed genes (>2 fold, p-value < 0.05) between parental and Δasp5 infected BMDMs, heatmaps were generated with genes selected from representative KEGG pathways. (Red: low, Green: high). Both up- and down-regulated genes of the host cell were analyzed together.
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Related In: Results  -  Collection

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

ppat.1005211.g006: ASP5 modulates host innate immune responses.(A) Interferon-γ stimulated, bone marrow derived macrophages (BMDMs) were infected with type I and type II parasite lines for 1 hr at a MOI of 1. In the absence of ASP5, type I parasites were not affected in IRG recruitment to the PVM. Type II parasites were used as controls. Parasites were labelled with α-GRA2, host IRGs with α-Irgb6 antibodies, and host nuclei with DAPI. IFAs are representative data from three independent biological experiments. Scale bars represent 2 μm. (B) IL-12p40 levels were measured by ELISA from supernatant collected 40 hr after BMDM infection with type I and type II strain parasites. Supernatant from uninfected cells was used as a control (u.i.). In both types, the absence of ASP5 dramatically reduces IL-12p40 levels. Data are mean value ± s.d. of three independent experiments. (*P<0.01, **P<0.005, Student’s t-test). (C) Quantitative chemokine production was determined by qRT-PCR on mouse embryonic fibroblast (pMEFs) infected with type I and type II parasites. Values were normalized to the amount of actin in each sample. Data are mean value ± s.d. of three independent experiments. (*P<0.01, **P<0.05, Student’s t-test). (D) Following KEGG analysis of the differentially expressed genes (>2 fold, p-value < 0.05) between parental and Δasp5 infected BMDMs, heatmaps were generated with genes selected from representative KEGG pathways. (Red: low, Green: high). Both up- and down-regulated genes of the host cell were analyzed together.
Mentions: One of the key events in establishing a protective Th1 immune response against T. gondii is the ability of host immune cells to produce the pro-inflammatory cytokine interleukin 12 (IL-12), which in turn stimulates the production of interferon gamma (IFNγ) by natural killer (NK) cells, CD4+ and CD8+ T cells [15, 24]. IFNγ is the major pro-inflammatory cytokine driving multiple cellular defense mechanisms during both the acute and chronic phases of infection [25]. Importantly, the immunity related GTPases (IRG proteins) constitute a large family of interferon-inducible proteins that mediate early resistance to T. gondii infection in mice. Several studies have shown that IRGs, in particular Irga6 and Irgb6 are recruited to the nascent PVM, where they cause disruption of the vacuole and parasite death. While the ROP18 complex of type I parasites is able to phosphorylate IRG proteins, thereby preventing their oligomerization and loading onto the PVM, type II parasites are unable to block the action of IRG proteins due to the polymorphic nature of ROP5, which forms part of the ROP18 complex [26]. Recent studies have associated GRA7 to the ROP18 complex by acting as regulator for ROP18-specific inactivation of Irga6 [23, 27]. To determine whether vacuoles containing RHΔasp5 parasites failed to block IRG recruitment to the PVM, an IRG recruitment assay was performed for Irgb6. Our results indicate that RHΔasp5 parasites behave like RH parasites and remain non-susceptible to Irgb6 and Irga6 loading (Fig 6A, left panel). Conversely, Irgb6 was recruited to the PVM of PRUΔku80Δasp5 parasites as previously reported for PRUΔku80 (Fig 6A, right panel) [26]. Given these data and in spite of the impact of ASP5 on GRA7 phosphorylation (which forms part of the ROP18 complex), we propose that ASP5 activity is not essential for the activity of the ROP18 complex [23].

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