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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

Synthesis scheme for generation of WEHI-586.Details of WEHI-586 synthesis is outlined in Materials and methods section. Reagents and conditions: a) N,N,N′,N′-Tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate,O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU), Et3N, dimethylformamide (DMF), HCl.NH2-Orn(N-Boc)-OMe; b) Pd/C, H2, MeOH; c) PhCH2SO2Cl, Et3N, dichloromethane (DCM); d) LiOH.H2O, tetrahydrofuran (THF), H2O; e) HBTU, Et3N, DMF, Ph(CH2)2NH2; f) 4N HCl, dioxane; g) HBTU, Et3N, DMF, 5; h) 4N HCl, dioxane; i) Et3N, N,N'-bis-Boc-1-guanylpyrazole; j) TFA, DCM.DOI:http://dx.doi.org/10.7554/eLife.10809.008
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fig2s4: Synthesis scheme for generation of WEHI-586.Details of WEHI-586 synthesis is outlined in Materials and methods section. Reagents and conditions: a) N,N,N′,N′-Tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate,O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU), Et3N, dimethylformamide (DMF), HCl.NH2-Orn(N-Boc)-OMe; b) Pd/C, H2, MeOH; c) PhCH2SO2Cl, Et3N, dichloromethane (DCM); d) LiOH.H2O, tetrahydrofuran (THF), H2O; e) HBTU, Et3N, DMF, Ph(CH2)2NH2; f) 4N HCl, dioxane; g) HBTU, Et3N, DMF, 5; h) 4N HCl, dioxane; i) Et3N, N,N'-bis-Boc-1-guanylpyrazole; j) TFA, DCM.DOI:http://dx.doi.org/10.7554/eLife.10809.008

Mentions: Compound numbers refer to the synthesis scheme outlined in Figure 2—figure supplement 4. A mixture of Cbz-Orn(N-Boc)-OH (500 mg, 1.36 mmol), Et3N (663 μL, 4.76 mmol), NH2Orn(N-Boc)-OMe. HCl (463 mg, 1.64 mmol), and HBTU (672 mg, 1.77 mmol), in DMF (5.0 mL) was allowed to stir for 18 hr at 20oC. 10% Citric acid solution was added to the reaction mixture. The solution was extracted with EtOAc (2 x 20 mL). The organic layer was then washed with 10% NaHCO3 solution (20 mL). The organic layer was dried (MgSO4) and the organic layer was concentrated in vacuo to obtain an oil. The oil obtained was subjected to silica chromatography gradient eluting with 100% DCM to 10% MeOH/DCM to obtain 1 as a white solid (630 mg, 78%). 1H NMR (CDCl3): δ 7.37 (s, 5H), 7.11 (br s, 1H), 5.60 (br s, 1H), 5.13 (s, 2H), 4.60–4.50 (m, 1H), 4.41–4.32 (m, 1H), 3.74 (s, 3H), 3.35–3.05 (m, 4H), 1.92–1.50 (m, 8H), 1.45 (s, 18H). MS, m/z = 595 [M+H]+.


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)

Synthesis scheme for generation of WEHI-586.Details of WEHI-586 synthesis is outlined in Materials and methods section. Reagents and conditions: a) N,N,N′,N′-Tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate,O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU), Et3N, dimethylformamide (DMF), HCl.NH2-Orn(N-Boc)-OMe; b) Pd/C, H2, MeOH; c) PhCH2SO2Cl, Et3N, dichloromethane (DCM); d) LiOH.H2O, tetrahydrofuran (THF), H2O; e) HBTU, Et3N, DMF, Ph(CH2)2NH2; f) 4N HCl, dioxane; g) HBTU, Et3N, DMF, 5; h) 4N HCl, dioxane; i) Et3N, N,N'-bis-Boc-1-guanylpyrazole; j) TFA, DCM.DOI:http://dx.doi.org/10.7554/eLife.10809.008
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4764566&req=5

fig2s4: Synthesis scheme for generation of WEHI-586.Details of WEHI-586 synthesis is outlined in Materials and methods section. Reagents and conditions: a) N,N,N′,N′-Tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate,O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU), Et3N, dimethylformamide (DMF), HCl.NH2-Orn(N-Boc)-OMe; b) Pd/C, H2, MeOH; c) PhCH2SO2Cl, Et3N, dichloromethane (DCM); d) LiOH.H2O, tetrahydrofuran (THF), H2O; e) HBTU, Et3N, DMF, Ph(CH2)2NH2; f) 4N HCl, dioxane; g) HBTU, Et3N, DMF, 5; h) 4N HCl, dioxane; i) Et3N, N,N'-bis-Boc-1-guanylpyrazole; j) TFA, DCM.DOI:http://dx.doi.org/10.7554/eLife.10809.008
Mentions: Compound numbers refer to the synthesis scheme outlined in Figure 2—figure supplement 4. A mixture of Cbz-Orn(N-Boc)-OH (500 mg, 1.36 mmol), Et3N (663 μL, 4.76 mmol), NH2Orn(N-Boc)-OMe. HCl (463 mg, 1.64 mmol), and HBTU (672 mg, 1.77 mmol), in DMF (5.0 mL) was allowed to stir for 18 hr at 20oC. 10% Citric acid solution was added to the reaction mixture. The solution was extracted with EtOAc (2 x 20 mL). The organic layer was then washed with 10% NaHCO3 solution (20 mL). The organic layer was dried (MgSO4) and the organic layer was concentrated in vacuo to obtain an oil. The oil obtained was subjected to silica chromatography gradient eluting with 100% DCM to 10% MeOH/DCM to obtain 1 as a white solid (630 mg, 78%). 1H NMR (CDCl3): δ 7.37 (s, 5H), 7.11 (br s, 1H), 5.60 (br s, 1H), 5.13 (s, 2H), 4.60–4.50 (m, 1H), 4.41–4.32 (m, 1H), 3.74 (s, 3H), 3.35–3.05 (m, 4H), 1.92–1.50 (m, 8H), 1.45 (s, 18H). MS, m/z = 595 [M+H]+.

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