Limits...
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 plays a major role in changing the host cell transcriptional response induced by Toxoplasma infection.(A) (i) Scatterplot of expression fold changes. The Y-axis shows log2-fold changes in HFFs infected with Δasp5 parasites versus uninfected HFFs (UI), while the X-axis shows log2-fold changes in HFFS infected with WT parasites (WT) vs. UI. The dashed line shows x=y. The solid line shows the least squares regression line through the origin. The regression has slope 0.6, showing that log fold changes for the Δasp5 parasites are only 60% of those for the wild-type parasites. Differentially expressed genes are color coded in the plot according to whether they change in both the WT and Δasp5 infections or only in the WT (false discovery rate < 0.05). Non-differentially expressed genes are shown in black. (ii) Numbers of genes corresponding to highlighted groups in the scatterplot. (B) Heat map of expression values for the 100 most differentially expressed genes for WT-infected HFFs versus uninfected. Z-scores are log2 counts per million, scaled to have mean 0 and standard deviation 1 for each gene. The plot shows that expression after Δasp5 infection tends to be intermediate between that of uninfected and WT-infected HFFs. (C) Barcode enrichment plot showing enrichment of Δgra16 regulated genes in the Δasp5 parasite infection expression changes. Genes are ordered from left to right in the plot from most up to most down during Δasp5 parasite infection. Specifically, genes are ranked from largest to smallest t-statistic for the Δasp5 versus WT comparison (X-axis). Genes up-regulated by Δgra16 versus WT in an independent experiment (Bougdour et al., 2013) are marked with vertical red bars. Similarly, genes down-regulated by Δgra16 in the independent experiment are marked with vertical blue bars. The worms show relative enrichment (Y-axes). The plot shows that Δasp5 up-regulated genes are strongly enriched for Δgra16 up-regulated genes (red) and Δasp5 down-regulated genes are strongly enriched for Δgra16 down-regulated genes (blue). HFFs, human foreskin fibroblasts; WT, wild type.DOI:http://dx.doi.org/10.7554/eLife.10809.017
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4764566&req=5

fig9: ASP5 plays a major role in changing the host cell transcriptional response induced by Toxoplasma infection.(A) (i) Scatterplot of expression fold changes. The Y-axis shows log2-fold changes in HFFs infected with Δasp5 parasites versus uninfected HFFs (UI), while the X-axis shows log2-fold changes in HFFS infected with WT parasites (WT) vs. UI. The dashed line shows x=y. The solid line shows the least squares regression line through the origin. The regression has slope 0.6, showing that log fold changes for the Δasp5 parasites are only 60% of those for the wild-type parasites. Differentially expressed genes are color coded in the plot according to whether they change in both the WT and Δasp5 infections or only in the WT (false discovery rate < 0.05). Non-differentially expressed genes are shown in black. (ii) Numbers of genes corresponding to highlighted groups in the scatterplot. (B) Heat map of expression values for the 100 most differentially expressed genes for WT-infected HFFs versus uninfected. Z-scores are log2 counts per million, scaled to have mean 0 and standard deviation 1 for each gene. The plot shows that expression after Δasp5 infection tends to be intermediate between that of uninfected and WT-infected HFFs. (C) Barcode enrichment plot showing enrichment of Δgra16 regulated genes in the Δasp5 parasite infection expression changes. Genes are ordered from left to right in the plot from most up to most down during Δasp5 parasite infection. Specifically, genes are ranked from largest to smallest t-statistic for the Δasp5 versus WT comparison (X-axis). Genes up-regulated by Δgra16 versus WT in an independent experiment (Bougdour et al., 2013) are marked with vertical red bars. Similarly, genes down-regulated by Δgra16 in the independent experiment are marked with vertical blue bars. The worms show relative enrichment (Y-axes). The plot shows that Δasp5 up-regulated genes are strongly enriched for Δgra16 up-regulated genes (red) and Δasp5 down-regulated genes are strongly enriched for Δgra16 down-regulated genes (blue). HFFs, human foreskin fibroblasts; WT, wild type.DOI:http://dx.doi.org/10.7554/eLife.10809.017

Mentions: Given our above findings, we wondered how important the ASP5-dependent pathway is to the transcriptional changes that Toxoplasma imparts on its host cell. Given that we determined there is little to no change in replication rates between WT and Δasp5 parasites (Figure 4B), we harvested all samples 20 hr after infection and used RNA sequencing (RNA-seq) to profile gene expression in HFFs that were either uninfected (UI), infected with WT (RH∆ku80) parasites, or infected with Δasp5 parasites. To make sure that all the changes that we observed were due to loss of ASP5 and not differences in tachyzoites numbers, we first compared the proportion of reads (rpkm) from parasite versus host cell origin as a readout of relative parasite numbers per sample. We saw equal amounts of reads mapping to human genes between all samples (24–27 × 106 reads), while infection with WT saw parasite RNA proportions of 27% (replicate 1), 24% (replicate 2) and 23% (replicate 3). Infection with our ASP5 deficient line saw parasite RNA to be 18% (replicate 1), 35% (replicate 2) and 36% (replicate 3) of the total reads, therefore suggesting that, in 2 out of the 3 samples, we have slightly more ASP5-deficient parasites per sample. Therefore, any loss of gene expression in ASP5-deficient cells must be due to loss of this protease and not lower amounts of overall parasites per sample. The expression changes due to infection by the Δasp5 parasites were generally smaller than those for the WT parasites. The log-fold change during infection with Δasp5 parasites was, on average, only 60% of the log-fold change during infection with WT parasites (Figure 9A-i). This suggests that most genes responding to parasite infection do so, at least partly, due to an ASP5-dependent pathway. At a false discovery rate of 5%, 3402 genes were significantly up-regulated and 3369 genes were significantly down-regulated in response to the WT infection, whereas only 1033 genes were significantly up- and 817 were significantly down-regulated in response to Δasp5 parasites. Of the 3402 genes up-regulated during WT infection, only 862 (25% ) remained significantly up-regulated upon deletion of ASP5 (Figure 9A-ii). Of the 3269 genes down-regulated during WT infection, only 742 (22%) remained significantly down-regulated upon deletion of ASP5 (Figure 9A-ii). This identifies genes (color-coded red and blue in Figure 9A) that are potentially unaffected by ASP5-dependent pathways.10.7554/eLife.10809.017Figure 9.ASP5 plays a major role in changing the host cell transcriptional response induced by Toxoplasma infection.


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 plays a major role in changing the host cell transcriptional response induced by Toxoplasma infection.(A) (i) Scatterplot of expression fold changes. The Y-axis shows log2-fold changes in HFFs infected with Δasp5 parasites versus uninfected HFFs (UI), while the X-axis shows log2-fold changes in HFFS infected with WT parasites (WT) vs. UI. The dashed line shows x=y. The solid line shows the least squares regression line through the origin. The regression has slope 0.6, showing that log fold changes for the Δasp5 parasites are only 60% of those for the wild-type parasites. Differentially expressed genes are color coded in the plot according to whether they change in both the WT and Δasp5 infections or only in the WT (false discovery rate < 0.05). Non-differentially expressed genes are shown in black. (ii) Numbers of genes corresponding to highlighted groups in the scatterplot. (B) Heat map of expression values for the 100 most differentially expressed genes for WT-infected HFFs versus uninfected. Z-scores are log2 counts per million, scaled to have mean 0 and standard deviation 1 for each gene. The plot shows that expression after Δasp5 infection tends to be intermediate between that of uninfected and WT-infected HFFs. (C) Barcode enrichment plot showing enrichment of Δgra16 regulated genes in the Δasp5 parasite infection expression changes. Genes are ordered from left to right in the plot from most up to most down during Δasp5 parasite infection. Specifically, genes are ranked from largest to smallest t-statistic for the Δasp5 versus WT comparison (X-axis). Genes up-regulated by Δgra16 versus WT in an independent experiment (Bougdour et al., 2013) are marked with vertical red bars. Similarly, genes down-regulated by Δgra16 in the independent experiment are marked with vertical blue bars. The worms show relative enrichment (Y-axes). The plot shows that Δasp5 up-regulated genes are strongly enriched for Δgra16 up-regulated genes (red) and Δasp5 down-regulated genes are strongly enriched for Δgra16 down-regulated genes (blue). HFFs, human foreskin fibroblasts; WT, wild type.DOI:http://dx.doi.org/10.7554/eLife.10809.017
© Copyright Policy
Related In: Results  -  Collection

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

fig9: ASP5 plays a major role in changing the host cell transcriptional response induced by Toxoplasma infection.(A) (i) Scatterplot of expression fold changes. The Y-axis shows log2-fold changes in HFFs infected with Δasp5 parasites versus uninfected HFFs (UI), while the X-axis shows log2-fold changes in HFFS infected with WT parasites (WT) vs. UI. The dashed line shows x=y. The solid line shows the least squares regression line through the origin. The regression has slope 0.6, showing that log fold changes for the Δasp5 parasites are only 60% of those for the wild-type parasites. Differentially expressed genes are color coded in the plot according to whether they change in both the WT and Δasp5 infections or only in the WT (false discovery rate < 0.05). Non-differentially expressed genes are shown in black. (ii) Numbers of genes corresponding to highlighted groups in the scatterplot. (B) Heat map of expression values for the 100 most differentially expressed genes for WT-infected HFFs versus uninfected. Z-scores are log2 counts per million, scaled to have mean 0 and standard deviation 1 for each gene. The plot shows that expression after Δasp5 infection tends to be intermediate between that of uninfected and WT-infected HFFs. (C) Barcode enrichment plot showing enrichment of Δgra16 regulated genes in the Δasp5 parasite infection expression changes. Genes are ordered from left to right in the plot from most up to most down during Δasp5 parasite infection. Specifically, genes are ranked from largest to smallest t-statistic for the Δasp5 versus WT comparison (X-axis). Genes up-regulated by Δgra16 versus WT in an independent experiment (Bougdour et al., 2013) are marked with vertical red bars. Similarly, genes down-regulated by Δgra16 in the independent experiment are marked with vertical blue bars. The worms show relative enrichment (Y-axes). The plot shows that Δasp5 up-regulated genes are strongly enriched for Δgra16 up-regulated genes (red) and Δasp5 down-regulated genes are strongly enriched for Δgra16 down-regulated genes (blue). HFFs, human foreskin fibroblasts; WT, wild type.DOI:http://dx.doi.org/10.7554/eLife.10809.017
Mentions: Given our above findings, we wondered how important the ASP5-dependent pathway is to the transcriptional changes that Toxoplasma imparts on its host cell. Given that we determined there is little to no change in replication rates between WT and Δasp5 parasites (Figure 4B), we harvested all samples 20 hr after infection and used RNA sequencing (RNA-seq) to profile gene expression in HFFs that were either uninfected (UI), infected with WT (RH∆ku80) parasites, or infected with Δasp5 parasites. To make sure that all the changes that we observed were due to loss of ASP5 and not differences in tachyzoites numbers, we first compared the proportion of reads (rpkm) from parasite versus host cell origin as a readout of relative parasite numbers per sample. We saw equal amounts of reads mapping to human genes between all samples (24–27 × 106 reads), while infection with WT saw parasite RNA proportions of 27% (replicate 1), 24% (replicate 2) and 23% (replicate 3). Infection with our ASP5 deficient line saw parasite RNA to be 18% (replicate 1), 35% (replicate 2) and 36% (replicate 3) of the total reads, therefore suggesting that, in 2 out of the 3 samples, we have slightly more ASP5-deficient parasites per sample. Therefore, any loss of gene expression in ASP5-deficient cells must be due to loss of this protease and not lower amounts of overall parasites per sample. The expression changes due to infection by the Δasp5 parasites were generally smaller than those for the WT parasites. The log-fold change during infection with Δasp5 parasites was, on average, only 60% of the log-fold change during infection with WT parasites (Figure 9A-i). This suggests that most genes responding to parasite infection do so, at least partly, due to an ASP5-dependent pathway. At a false discovery rate of 5%, 3402 genes were significantly up-regulated and 3369 genes were significantly down-regulated in response to the WT infection, whereas only 1033 genes were significantly up- and 817 were significantly down-regulated in response to Δasp5 parasites. Of the 3402 genes up-regulated during WT infection, only 862 (25% ) remained significantly up-regulated upon deletion of ASP5 (Figure 9A-ii). Of the 3269 genes down-regulated during WT infection, only 742 (22%) remained significantly down-regulated upon deletion of ASP5 (Figure 9A-ii). This identifies genes (color-coded red and blue in Figure 9A) that are potentially unaffected by ASP5-dependent pathways.10.7554/eLife.10809.017Figure 9.ASP5 plays a major role in changing the host cell transcriptional response induced by Toxoplasma infection.

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