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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

ASP5 influences efficient host mitochondrial recruitment and assembly of the NTN.(A) Electron micrographs of intracellular WT (Δku80) (i and iii) and Δasp5 (ii and iv) tachyzoites within HFFs. Bars represent 1 µm (i,ii) and 200 nm (iii, iv). (i, ii) Low-power image showing WT (i) and Δasp5 (ii) tachyzoites containing a nucleus (N), rhoptries (R), micronemes (M), dense granules (D) and a Golgi body (G) located within a PV. Note the large number of host cell mitochondria (arrowheads) associated with the PVM and the large NTN within the PV in wild-type parasites compared to Δasp5 parasites. (iii, iv) Details from the periphery of the PV showing a large host cell mitochondrion (HM) closely applied to the PVM in the wild type (iii) compared to the smaller mitochondrion (HM) associated with the Δasp5 PV (iv). (B) Quantitation of percentage of the PVM associated with host mitochondria, 5.59 ± 2.08% for Δasp5 parasites versus 24.3 ± 6.98% for wild-type parasites, mean ± standard error of the mean, P < 0.0001, n = 20 vacuoles. (C) (i) Mouse embryonic fibroblasts expressing MTS-GFP infected for 4 hr with wild type (Δhx), Δasp5CRISPR (a non-GFP positive knock out) or two independent ASP5 complemented clones (Δasp5CRISPR:ASP5WT-HA3). Localization of MAF1 at the PVM (top panel and bottom two panels) and mislocalized in intraparasitic puncta, potentially dense granules (panels 2 and 5), are shown in red. Mitochondria (MTS-GFP) are localized at the PVM in wild-type parasites (panel 1) and Δasp5CRISPR:ASP5WT-HA3 clones 1 and 2 (panels 5–6) to a large extent, but less so in the Δasp5CRISPR parasites (panels 2–4). (ii) Immunoblot using αHA antibodies against parasites expressing ASP5WT-HA3 and complemented mutants Δasp5CRISPR:ASP5WT-HA3 clones 1 and 2 shows the parasites express similar levels of HA-tagged ASP5 (as in Figure 2A), αGAP45 serves as a loading control. (D) Western blot of MAF1 species in wild-type and Δku80Δasp5 parasites. Blue arrow shows non-specific labeling (NS), αCatalase serves as a loading control. (E) Electron micrographs of intracellular wild type (i and ii) and Δku80Δasp5 (iii and iv) tachyzoites. Bars represent 1 µm (i, iii) and 200 nm (ii, iv). (i, ii) Low-power image showing wild-type (i) and Δasp5 (iii) tachyzoites containing a nucleus (N), rhoptries (R), micronemes (M), and dense granules (D) located within the PV. The large number of host cell mitochondria (arrowheads) associated with the PVM and the large NTN within the PV in the wild type compared to the Δasp5 parasites is noteworthy. (ii) Detail of the PV of a WT parasite showing the intertwining tubules of the NTN. HM – host cell mitochondrion. (iv) Detail of the PV surrounding a Δasp5 parasite showing granular material and a few vesicles (V) but absence of the tubular network. HM – host cell mitochondrion. Scale bar is 5 μm. ASP5, Aspartyl Protease 5; GFP, green fluorescent protein; HFFs, human foreskin fibroblasts; MTS, mitochondrial targeting sequence; NTN, nanotubular network; PV, parasitophorous vacuole; PVM, parasitophorous vacuole membrane; WT, wild type.DOI:http://dx.doi.org/10.7554/eLife.10809.014
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fig7: ASP5 influences efficient host mitochondrial recruitment and assembly of the NTN.(A) Electron micrographs of intracellular WT (Δku80) (i and iii) and Δasp5 (ii and iv) tachyzoites within HFFs. Bars represent 1 µm (i,ii) and 200 nm (iii, iv). (i, ii) Low-power image showing WT (i) and Δasp5 (ii) tachyzoites containing a nucleus (N), rhoptries (R), micronemes (M), dense granules (D) and a Golgi body (G) located within a PV. Note the large number of host cell mitochondria (arrowheads) associated with the PVM and the large NTN within the PV in wild-type parasites compared to Δasp5 parasites. (iii, iv) Details from the periphery of the PV showing a large host cell mitochondrion (HM) closely applied to the PVM in the wild type (iii) compared to the smaller mitochondrion (HM) associated with the Δasp5 PV (iv). (B) Quantitation of percentage of the PVM associated with host mitochondria, 5.59 ± 2.08% for Δasp5 parasites versus 24.3 ± 6.98% for wild-type parasites, mean ± standard error of the mean, P < 0.0001, n = 20 vacuoles. (C) (i) Mouse embryonic fibroblasts expressing MTS-GFP infected for 4 hr with wild type (Δhx), Δasp5CRISPR (a non-GFP positive knock out) or two independent ASP5 complemented clones (Δasp5CRISPR:ASP5WT-HA3). Localization of MAF1 at the PVM (top panel and bottom two panels) and mislocalized in intraparasitic puncta, potentially dense granules (panels 2 and 5), are shown in red. Mitochondria (MTS-GFP) are localized at the PVM in wild-type parasites (panel 1) and Δasp5CRISPR:ASP5WT-HA3 clones 1 and 2 (panels 5–6) to a large extent, but less so in the Δasp5CRISPR parasites (panels 2–4). (ii) Immunoblot using αHA antibodies against parasites expressing ASP5WT-HA3 and complemented mutants Δasp5CRISPR:ASP5WT-HA3 clones 1 and 2 shows the parasites express similar levels of HA-tagged ASP5 (as in Figure 2A), αGAP45 serves as a loading control. (D) Western blot of MAF1 species in wild-type and Δku80Δasp5 parasites. Blue arrow shows non-specific labeling (NS), αCatalase serves as a loading control. (E) Electron micrographs of intracellular wild type (i and ii) and Δku80Δasp5 (iii and iv) tachyzoites. Bars represent 1 µm (i, iii) and 200 nm (ii, iv). (i, ii) Low-power image showing wild-type (i) and Δasp5 (iii) tachyzoites containing a nucleus (N), rhoptries (R), micronemes (M), and dense granules (D) located within the PV. The large number of host cell mitochondria (arrowheads) associated with the PVM and the large NTN within the PV in the wild type compared to the Δasp5 parasites is noteworthy. (ii) Detail of the PV of a WT parasite showing the intertwining tubules of the NTN. HM – host cell mitochondrion. (iv) Detail of the PV surrounding a Δasp5 parasite showing granular material and a few vesicles (V) but absence of the tubular network. HM – host cell mitochondrion. Scale bar is 5 μm. ASP5, Aspartyl Protease 5; GFP, green fluorescent protein; HFFs, human foreskin fibroblasts; MTS, mitochondrial targeting sequence; NTN, nanotubular network; PV, parasitophorous vacuole; PVM, parasitophorous vacuole membrane; WT, wild type.DOI:http://dx.doi.org/10.7554/eLife.10809.014

Mentions: (A) (i) Schematic representation of the ASP5 knockout strategy in the RHΔku80:GRA16-HA line using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 and plasmid-based recombination. (ii) PCR confirmation of a resulting Δku80Δasp5 clonal line. Sequencing of PCR products confirmed atypical integration topology (data not shown). (B) Generation of the Δasp5CRISPR parasites, where parental RHΔhx tachyzoites were transfected with pU6-Universal-mCherry-sgASP5-2 (see Materials and methods) and a clone was chosen with an insertion of ‘TT’ at the predicted Cas9 cleavage site, resulting in a frameshift mutation in the coding region of ASP5. (C) Following transfection, two clones were chosen with stably-integrated ASP5WT-HA3 in the Δasp5CRISPR line, generating the Δasp5CRISPR:ASP5WT-HA3 parasites. Δasp5CRISPR:ASP5WT-HA3 is exclusively used to refer to clone 1 in this manuscript, with the exception of Figure 7C, where both clones are used. ASP5, Aspartyl Protease 5; HA, hemagglutinin; HA3, triple-hemagglutinin; PCR, polymerase chain reaction.


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)

ASP5 influences efficient host mitochondrial recruitment and assembly of the NTN.(A) Electron micrographs of intracellular WT (Δku80) (i and iii) and Δasp5 (ii and iv) tachyzoites within HFFs. Bars represent 1 µm (i,ii) and 200 nm (iii, iv). (i, ii) Low-power image showing WT (i) and Δasp5 (ii) tachyzoites containing a nucleus (N), rhoptries (R), micronemes (M), dense granules (D) and a Golgi body (G) located within a PV. Note the large number of host cell mitochondria (arrowheads) associated with the PVM and the large NTN within the PV in wild-type parasites compared to Δasp5 parasites. (iii, iv) Details from the periphery of the PV showing a large host cell mitochondrion (HM) closely applied to the PVM in the wild type (iii) compared to the smaller mitochondrion (HM) associated with the Δasp5 PV (iv). (B) Quantitation of percentage of the PVM associated with host mitochondria, 5.59 ± 2.08% for Δasp5 parasites versus 24.3 ± 6.98% for wild-type parasites, mean ± standard error of the mean, P < 0.0001, n = 20 vacuoles. (C) (i) Mouse embryonic fibroblasts expressing MTS-GFP infected for 4 hr with wild type (Δhx), Δasp5CRISPR (a non-GFP positive knock out) or two independent ASP5 complemented clones (Δasp5CRISPR:ASP5WT-HA3). Localization of MAF1 at the PVM (top panel and bottom two panels) and mislocalized in intraparasitic puncta, potentially dense granules (panels 2 and 5), are shown in red. Mitochondria (MTS-GFP) are localized at the PVM in wild-type parasites (panel 1) and Δasp5CRISPR:ASP5WT-HA3 clones 1 and 2 (panels 5–6) to a large extent, but less so in the Δasp5CRISPR parasites (panels 2–4). (ii) Immunoblot using αHA antibodies against parasites expressing ASP5WT-HA3 and complemented mutants Δasp5CRISPR:ASP5WT-HA3 clones 1 and 2 shows the parasites express similar levels of HA-tagged ASP5 (as in Figure 2A), αGAP45 serves as a loading control. (D) Western blot of MAF1 species in wild-type and Δku80Δasp5 parasites. Blue arrow shows non-specific labeling (NS), αCatalase serves as a loading control. (E) Electron micrographs of intracellular wild type (i and ii) and Δku80Δasp5 (iii and iv) tachyzoites. Bars represent 1 µm (i, iii) and 200 nm (ii, iv). (i, ii) Low-power image showing wild-type (i) and Δasp5 (iii) tachyzoites containing a nucleus (N), rhoptries (R), micronemes (M), and dense granules (D) located within the PV. The large number of host cell mitochondria (arrowheads) associated with the PVM and the large NTN within the PV in the wild type compared to the Δasp5 parasites is noteworthy. (ii) Detail of the PV of a WT parasite showing the intertwining tubules of the NTN. HM – host cell mitochondrion. (iv) Detail of the PV surrounding a Δasp5 parasite showing granular material and a few vesicles (V) but absence of the tubular network. HM – host cell mitochondrion. Scale bar is 5 μm. ASP5, Aspartyl Protease 5; GFP, green fluorescent protein; HFFs, human foreskin fibroblasts; MTS, mitochondrial targeting sequence; NTN, nanotubular network; PV, parasitophorous vacuole; PVM, parasitophorous vacuole membrane; WT, wild type.DOI:http://dx.doi.org/10.7554/eLife.10809.014
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fig7: ASP5 influences efficient host mitochondrial recruitment and assembly of the NTN.(A) Electron micrographs of intracellular WT (Δku80) (i and iii) and Δasp5 (ii and iv) tachyzoites within HFFs. Bars represent 1 µm (i,ii) and 200 nm (iii, iv). (i, ii) Low-power image showing WT (i) and Δasp5 (ii) tachyzoites containing a nucleus (N), rhoptries (R), micronemes (M), dense granules (D) and a Golgi body (G) located within a PV. Note the large number of host cell mitochondria (arrowheads) associated with the PVM and the large NTN within the PV in wild-type parasites compared to Δasp5 parasites. (iii, iv) Details from the periphery of the PV showing a large host cell mitochondrion (HM) closely applied to the PVM in the wild type (iii) compared to the smaller mitochondrion (HM) associated with the Δasp5 PV (iv). (B) Quantitation of percentage of the PVM associated with host mitochondria, 5.59 ± 2.08% for Δasp5 parasites versus 24.3 ± 6.98% for wild-type parasites, mean ± standard error of the mean, P < 0.0001, n = 20 vacuoles. (C) (i) Mouse embryonic fibroblasts expressing MTS-GFP infected for 4 hr with wild type (Δhx), Δasp5CRISPR (a non-GFP positive knock out) or two independent ASP5 complemented clones (Δasp5CRISPR:ASP5WT-HA3). Localization of MAF1 at the PVM (top panel and bottom two panels) and mislocalized in intraparasitic puncta, potentially dense granules (panels 2 and 5), are shown in red. Mitochondria (MTS-GFP) are localized at the PVM in wild-type parasites (panel 1) and Δasp5CRISPR:ASP5WT-HA3 clones 1 and 2 (panels 5–6) to a large extent, but less so in the Δasp5CRISPR parasites (panels 2–4). (ii) Immunoblot using αHA antibodies against parasites expressing ASP5WT-HA3 and complemented mutants Δasp5CRISPR:ASP5WT-HA3 clones 1 and 2 shows the parasites express similar levels of HA-tagged ASP5 (as in Figure 2A), αGAP45 serves as a loading control. (D) Western blot of MAF1 species in wild-type and Δku80Δasp5 parasites. Blue arrow shows non-specific labeling (NS), αCatalase serves as a loading control. (E) Electron micrographs of intracellular wild type (i and ii) and Δku80Δasp5 (iii and iv) tachyzoites. Bars represent 1 µm (i, iii) and 200 nm (ii, iv). (i, ii) Low-power image showing wild-type (i) and Δasp5 (iii) tachyzoites containing a nucleus (N), rhoptries (R), micronemes (M), and dense granules (D) located within the PV. The large number of host cell mitochondria (arrowheads) associated with the PVM and the large NTN within the PV in the wild type compared to the Δasp5 parasites is noteworthy. (ii) Detail of the PV of a WT parasite showing the intertwining tubules of the NTN. HM – host cell mitochondrion. (iv) Detail of the PV surrounding a Δasp5 parasite showing granular material and a few vesicles (V) but absence of the tubular network. HM – host cell mitochondrion. Scale bar is 5 μm. ASP5, Aspartyl Protease 5; GFP, green fluorescent protein; HFFs, human foreskin fibroblasts; MTS, mitochondrial targeting sequence; NTN, nanotubular network; PV, parasitophorous vacuole; PVM, parasitophorous vacuole membrane; WT, wild type.DOI:http://dx.doi.org/10.7554/eLife.10809.014
Mentions: (A) (i) Schematic representation of the ASP5 knockout strategy in the RHΔku80:GRA16-HA line using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 and plasmid-based recombination. (ii) PCR confirmation of a resulting Δku80Δasp5 clonal line. Sequencing of PCR products confirmed atypical integration topology (data not shown). (B) Generation of the Δasp5CRISPR parasites, where parental RHΔhx tachyzoites were transfected with pU6-Universal-mCherry-sgASP5-2 (see Materials and methods) and a clone was chosen with an insertion of ‘TT’ at the predicted Cas9 cleavage site, resulting in a frameshift mutation in the coding region of ASP5. (C) Following transfection, two clones were chosen with stably-integrated ASP5WT-HA3 in the Δasp5CRISPR line, generating the Δasp5CRISPR:ASP5WT-HA3 parasites. Δasp5CRISPR:ASP5WT-HA3 is exclusively used to refer to clone 1 in this manuscript, with the exception of Figure 7C, where both clones are used. ASP5, Aspartyl Protease 5; HA, hemagglutinin; HA3, triple-hemagglutinin; PCR, polymerase chain reaction.

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