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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

Complementation of Δasp5 parasites restores export of GRA24.Localisation of transiently transfected GRA24-Myc3 of in both Δasp5CRISPR and Δasp5CRISPR:ASP5WT-HA3 (Clone 1) tachyzoites. Filled arrowheads signify the position of host nuclei (DAPI), open arrowheads identify non-transfected parasites and GAP45 marks the periphery of tachyzoites. Scale bar is 5 μm. DAPI, 4',6-diamidino-2-phenylindole; HA3, triple-hemagglutininDOI:http://dx.doi.org/10.7554/eLife.10809.016
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fig8s1: Complementation of Δasp5 parasites restores export of GRA24.Localisation of transiently transfected GRA24-Myc3 of in both Δasp5CRISPR and Δasp5CRISPR:ASP5WT-HA3 (Clone 1) tachyzoites. Filled arrowheads signify the position of host nuclei (DAPI), open arrowheads identify non-transfected parasites and GAP45 marks the periphery of tachyzoites. Scale bar is 5 μm. DAPI, 4',6-diamidino-2-phenylindole; HA3, triple-hemagglutininDOI:http://dx.doi.org/10.7554/eLife.10809.016

Mentions: Following the identification of GRA24 as an exported effector protein that traffics to the host nucleus (Braun et al., 2013), we sought to determine whether its translocation into the host cell is also ASP5-dependent. WT and Δasp5 parasites were transfected with an ectopic copy of GRA24 fused to 3xMyc tags (GRA24-Myc3), which was integrated into the uracil phosphoribosyltransferase (URPT) locus. GRA24-Myc3 was expressed in parasites and exported to the host cell nucleus by WT parasites as previously described (Braun et al., 2013); however, export was completely blocked in Δasp5 parasites (Figure 8A, Figure 8—figure supplement 1). Complementation of Δasp5 parasites with ASP5WT-HA3 restored the export of GRA24-Myc3 (Figure 8—figure supplement 1). Despite the requirement of ASP5 for GRA24 export, assessment of processing by Western blot did not reveal any size difference in GRA24-Myc3 between WT and Δasp5 parasites (Figure 8B). While GRA24 lacks a canonical TEXEL sequence (RRLxx), it does contain the non-canonical TEXEL-like sequences RGYHG, RGGLQ and RSLGM, and so we assessed whether these might be cleaved by ASP5 using synthetic peptides; however, none were efficiently processed (Figure 8C). Collectively, this suggests that GRA24 is not a direct substrate of ASP5 but its export is dependent on this protease.10.7554/eLife.10809.015Figure 8.GRA24 requires ASP5 for export but not processing.


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)

Complementation of Δasp5 parasites restores export of GRA24.Localisation of transiently transfected GRA24-Myc3 of in both Δasp5CRISPR and Δasp5CRISPR:ASP5WT-HA3 (Clone 1) tachyzoites. Filled arrowheads signify the position of host nuclei (DAPI), open arrowheads identify non-transfected parasites and GAP45 marks the periphery of tachyzoites. Scale bar is 5 μm. DAPI, 4',6-diamidino-2-phenylindole; HA3, triple-hemagglutininDOI:http://dx.doi.org/10.7554/eLife.10809.016
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4764566&req=5

fig8s1: Complementation of Δasp5 parasites restores export of GRA24.Localisation of transiently transfected GRA24-Myc3 of in both Δasp5CRISPR and Δasp5CRISPR:ASP5WT-HA3 (Clone 1) tachyzoites. Filled arrowheads signify the position of host nuclei (DAPI), open arrowheads identify non-transfected parasites and GAP45 marks the periphery of tachyzoites. Scale bar is 5 μm. DAPI, 4',6-diamidino-2-phenylindole; HA3, triple-hemagglutininDOI:http://dx.doi.org/10.7554/eLife.10809.016
Mentions: Following the identification of GRA24 as an exported effector protein that traffics to the host nucleus (Braun et al., 2013), we sought to determine whether its translocation into the host cell is also ASP5-dependent. WT and Δasp5 parasites were transfected with an ectopic copy of GRA24 fused to 3xMyc tags (GRA24-Myc3), which was integrated into the uracil phosphoribosyltransferase (URPT) locus. GRA24-Myc3 was expressed in parasites and exported to the host cell nucleus by WT parasites as previously described (Braun et al., 2013); however, export was completely blocked in Δasp5 parasites (Figure 8A, Figure 8—figure supplement 1). Complementation of Δasp5 parasites with ASP5WT-HA3 restored the export of GRA24-Myc3 (Figure 8—figure supplement 1). Despite the requirement of ASP5 for GRA24 export, assessment of processing by Western blot did not reveal any size difference in GRA24-Myc3 between WT and Δasp5 parasites (Figure 8B). While GRA24 lacks a canonical TEXEL sequence (RRLxx), it does contain the non-canonical TEXEL-like sequences RGYHG, RGGLQ and RSLGM, and so we assessed whether these might be cleaved by ASP5 using synthetic peptides; however, none were efficiently processed (Figure 8C). Collectively, this suggests that GRA24 is not a direct substrate of ASP5 but its export is dependent on this protease.10.7554/eLife.10809.015Figure 8.GRA24 requires ASP5 for export but not processing.

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