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An aspartyl protease defines a novel pathway for export of Toxoplasma proteins into the host cell.

Coffey MJ, Sleebs BE, Uboldi AD, Garnham A, Franco M, Marino ND, Panas MW, Ferguson DJ, Enciso M, O'Neill MT, Lopaticki S, Stewart RJ, Dewson G, Smyth GK, Smith BJ, Masters SL, Boothroyd JC, Boddey JA, Tonkin CJ - Elife (2015)

Bottom Line: Here, we identify a novel host cell effector export pathway that requires the Golgi-resident aspartyl protease 5 (ASP5).All these changes result in attenuation of virulence of Δasp5 tachyzoites in vivo.This work characterizes the first identified machinery required for export of Toxoplasma effectors into the infected host cell.

View Article: PubMed Central - PubMed

Affiliation: The Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia.

ABSTRACT
Infection by Toxoplasma gondii leads to massive changes to the host cell. Here, we identify a novel host cell effector export pathway that requires the Golgi-resident aspartyl protease 5 (ASP5). We demonstrate that ASP5 cleaves a highly constrained amino acid motif that has similarity to the PEXEL-motif of Plasmodium parasites. We show that ASP5 matures substrates at both the N- and C-terminal ends of proteins and also controls trafficking of effectors without this motif. Furthermore, ASP5 controls establishment of the nanotubular network and is required for the efficient recruitment of host mitochondria to the vacuole. Assessment of host gene expression reveals that the ASP5-dependent pathway influences thousands of the transcriptional changes that Toxoplasma imparts on its host cell. All these changes result in attenuation of virulence of Δasp5 tachyzoites in vivo. This work characterizes the first identified machinery required for export of Toxoplasma effectors into the infected host cell.

No MeSH data available.


Related in: MedlinePlus

Induction of host c-Myc is ASP5-dependent.(A) Representative IFAs 14 hr after infection of c-Myc expression in confluent HFFs. Mock-infected cells express very little c-Myc while infection with WT parasites leads to a dramatic up-regulation of this transcription factor. Δasp5CRISPR-infected cells express marginally more c-Myc than mock-infected, which is complemented by the re-introduction of ASP5 (Δasp5CRISPR:ASP5WT-HA3). (B) Quantitation of c-Myc signal (as a ratio of DAPI signal) in cells from (A), P = 0.0001, values are mean ± standard deviation, unpaired t-test, n ≥ 20 nuclei from cells infected with single vacuoles. (C) Western blot showing up-regulation of c-Myc upon wild type infection, which is drastically decreased following deletion of ASP5. αSAG1 and αGAPDH serve as parasite and host loading controls, respectively. Scale bars are 20 μm. HA3, triple-hemagglutinin; HFFs, human foreskin fibroblasts; IFA, immunofluorescence assay; WT, wild type.DOI:http://dx.doi.org/10.7554/eLife.10809.012
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fig5: Induction of host c-Myc is ASP5-dependent.(A) Representative IFAs 14 hr after infection of c-Myc expression in confluent HFFs. Mock-infected cells express very little c-Myc while infection with WT parasites leads to a dramatic up-regulation of this transcription factor. Δasp5CRISPR-infected cells express marginally more c-Myc than mock-infected, which is complemented by the re-introduction of ASP5 (Δasp5CRISPR:ASP5WT-HA3). (B) Quantitation of c-Myc signal (as a ratio of DAPI signal) in cells from (A), P = 0.0001, values are mean ± standard deviation, unpaired t-test, n ≥ 20 nuclei from cells infected with single vacuoles. (C) Western blot showing up-regulation of c-Myc upon wild type infection, which is drastically decreased following deletion of ASP5. αSAG1 and αGAPDH serve as parasite and host loading controls, respectively. Scale bars are 20 μm. HA3, triple-hemagglutinin; HFFs, human foreskin fibroblasts; IFA, immunofluorescence assay; WT, wild type.DOI:http://dx.doi.org/10.7554/eLife.10809.012

Mentions: We sought to determine the importance of ASP5 in controlling other cellular phenotypes that Toxoplasma imparts on its host cell. It has recently been shown that Toxoplasma tachyzoites, but not the related Neospora species, actively induce expression of host c-Myc following infection (Franco et al., 2014). This activation of c-Myc was not induced in response to parasite invasion or injection of rhoptry proteins (Franco et al., 2014), suggesting that one or more dense granule proteins may be responsible. To determine whether ASP5 is involved in up-regulation of host c-Myc, HFFs were infected with WT, Δasp5 or Δasp5CRISPR:ASP5WT-HA3 parasites, and c-Myc expression was measured by IFA and immunoblot. While uninfected HFFs showed little c-Myc expression by IFA, cells infected with WT Toxoplasma, or parasites with complemented ASP5 expression, had almost universal induction of c-Myc in their nuclei, as previously reported (Figure 5A) (Franco et al., 2014). Upon infection with Δasp5 parasites, a sharp reduction in c-Myc expression within the nuclei was observed (Figure 5A). Quantification by IFA showed that HFFs infected with parasites lacking ASP5 expressed approximately 6.4-fold less c-Myc than those infected with WT parasites (normalized ratio of c-Myc to 4',6-diamidino-2-phenylindole [DAPI]) (Figure 5B). To confirm this, c-Myc induction was measured by immunoblot of whole cell protein fractions. While c-Myc expression was induced in host cells infected with WT parasites, the signal was dramatically reduced in HFFs infected with the same number of Δasp5 parasites (Figure 5C), confirming that ASP5 is required for Toxoplasma to induce c-Myc in infected cells. Together, this work suggests that the up-regulation of c-Myc induced by tachyzoites is controlled by one or more ASP5-dependent proteins.10.7554/eLife.10809.012Figure 5.Induction of host c-Myc is ASP5-dependent.


An aspartyl protease defines a novel pathway for export of Toxoplasma proteins into the host cell.

Coffey MJ, Sleebs BE, Uboldi AD, Garnham A, Franco M, Marino ND, Panas MW, Ferguson DJ, Enciso M, O'Neill MT, Lopaticki S, Stewart RJ, Dewson G, Smyth GK, Smith BJ, Masters SL, Boothroyd JC, Boddey JA, Tonkin CJ - Elife (2015)

Induction of host c-Myc is ASP5-dependent.(A) Representative IFAs 14 hr after infection of c-Myc expression in confluent HFFs. Mock-infected cells express very little c-Myc while infection with WT parasites leads to a dramatic up-regulation of this transcription factor. Δasp5CRISPR-infected cells express marginally more c-Myc than mock-infected, which is complemented by the re-introduction of ASP5 (Δasp5CRISPR:ASP5WT-HA3). (B) Quantitation of c-Myc signal (as a ratio of DAPI signal) in cells from (A), P = 0.0001, values are mean ± standard deviation, unpaired t-test, n ≥ 20 nuclei from cells infected with single vacuoles. (C) Western blot showing up-regulation of c-Myc upon wild type infection, which is drastically decreased following deletion of ASP5. αSAG1 and αGAPDH serve as parasite and host loading controls, respectively. Scale bars are 20 μm. HA3, triple-hemagglutinin; HFFs, human foreskin fibroblasts; IFA, immunofluorescence assay; WT, wild type.DOI:http://dx.doi.org/10.7554/eLife.10809.012
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Related In: Results  -  Collection

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fig5: Induction of host c-Myc is ASP5-dependent.(A) Representative IFAs 14 hr after infection of c-Myc expression in confluent HFFs. Mock-infected cells express very little c-Myc while infection with WT parasites leads to a dramatic up-regulation of this transcription factor. Δasp5CRISPR-infected cells express marginally more c-Myc than mock-infected, which is complemented by the re-introduction of ASP5 (Δasp5CRISPR:ASP5WT-HA3). (B) Quantitation of c-Myc signal (as a ratio of DAPI signal) in cells from (A), P = 0.0001, values are mean ± standard deviation, unpaired t-test, n ≥ 20 nuclei from cells infected with single vacuoles. (C) Western blot showing up-regulation of c-Myc upon wild type infection, which is drastically decreased following deletion of ASP5. αSAG1 and αGAPDH serve as parasite and host loading controls, respectively. Scale bars are 20 μm. HA3, triple-hemagglutinin; HFFs, human foreskin fibroblasts; IFA, immunofluorescence assay; WT, wild type.DOI:http://dx.doi.org/10.7554/eLife.10809.012
Mentions: We sought to determine the importance of ASP5 in controlling other cellular phenotypes that Toxoplasma imparts on its host cell. It has recently been shown that Toxoplasma tachyzoites, but not the related Neospora species, actively induce expression of host c-Myc following infection (Franco et al., 2014). This activation of c-Myc was not induced in response to parasite invasion or injection of rhoptry proteins (Franco et al., 2014), suggesting that one or more dense granule proteins may be responsible. To determine whether ASP5 is involved in up-regulation of host c-Myc, HFFs were infected with WT, Δasp5 or Δasp5CRISPR:ASP5WT-HA3 parasites, and c-Myc expression was measured by IFA and immunoblot. While uninfected HFFs showed little c-Myc expression by IFA, cells infected with WT Toxoplasma, or parasites with complemented ASP5 expression, had almost universal induction of c-Myc in their nuclei, as previously reported (Figure 5A) (Franco et al., 2014). Upon infection with Δasp5 parasites, a sharp reduction in c-Myc expression within the nuclei was observed (Figure 5A). Quantification by IFA showed that HFFs infected with parasites lacking ASP5 expressed approximately 6.4-fold less c-Myc than those infected with WT parasites (normalized ratio of c-Myc to 4',6-diamidino-2-phenylindole [DAPI]) (Figure 5B). To confirm this, c-Myc induction was measured by immunoblot of whole cell protein fractions. While c-Myc expression was induced in host cells infected with WT parasites, the signal was dramatically reduced in HFFs infected with the same number of Δasp5 parasites (Figure 5C), confirming that ASP5 is required for Toxoplasma to induce c-Myc in infected cells. Together, this work suggests that the up-regulation of c-Myc induced by tachyzoites is controlled by one or more ASP5-dependent proteins.10.7554/eLife.10809.012Figure 5.Induction of host c-Myc is ASP5-dependent.

Bottom Line: Here, we identify a novel host cell effector export pathway that requires the Golgi-resident aspartyl protease 5 (ASP5).All these changes result in attenuation of virulence of Δasp5 tachyzoites in vivo.This work characterizes the first identified machinery required for export of Toxoplasma effectors into the infected host cell.

View Article: PubMed Central - PubMed

Affiliation: The Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia.

ABSTRACT
Infection by Toxoplasma gondii leads to massive changes to the host cell. Here, we identify a novel host cell effector export pathway that requires the Golgi-resident aspartyl protease 5 (ASP5). We demonstrate that ASP5 cleaves a highly constrained amino acid motif that has similarity to the PEXEL-motif of Plasmodium parasites. We show that ASP5 matures substrates at both the N- and C-terminal ends of proteins and also controls trafficking of effectors without this motif. Furthermore, ASP5 controls establishment of the nanotubular network and is required for the efficient recruitment of host mitochondria to the vacuole. Assessment of host gene expression reveals that the ASP5-dependent pathway influences thousands of the transcriptional changes that Toxoplasma imparts on its host cell. All these changes result in attenuation of virulence of Δasp5 tachyzoites in vivo. This work characterizes the first identified machinery required for export of Toxoplasma effectors into the infected host cell.

No MeSH data available.


Related in: MedlinePlus