Figure 1.
TgGCN5-A mRNA is up-regulated in response to alkaline stress.
Wild-type (WT) parasites were incubated in control (pH 7.0) or alkaline media (pH 8.2) for three days. The levels of mRNA expression for TgGCN5-A or ACT1 were monitored by quantitative real-time PCR. Gene expression relative to control is shown. Error bars indicate the S.D.
Figure 2.
Genes modulated during alkaline pH stress in wild-type Toxoplasma.
Microarray analysis was performed on wild-type parasites incubated in HFFs cells under normal culture conditions (pH 7.0) or alkaline conditions (pH 8.2) for three days. Pie chart displays the GO categories of genes reported to be (A) up-regulated or (B) down-regulated in response to alkaline conditions (p<0.001).
Table 1.
Predicted zinc finger proteins up-regulated during alkaline stress in Toxoplasma.
Table 2.
Predicted AP2 proteins up-regulated during alkaline stress in Toxoplasma.
Figure 3.
ΔGCN5-A parasites fail to up-regulate alkaline pH response genes.
A. Scatter plot of expression levels for ΔGCN5-A (KO – vertical axis) vs. wild-type (WT –horizontal axis) under normal conditions. Data limited to those genes with fraction present ≥0.5 in WT or KO. B. Scatter plot of wild-type (WT) vs. ΔGCN5-A knockout (KO) fold changes. Probe sets limited to those with maximum fraction present ≥0.5, p<0.01 and fold ≥2.0 for WT stress vs. normal culture conditions. Regression line is in black. All points below the red line are genes with smaller fold changes in KO than in WT for stress vs. normal culture conditions.
Figure 4.
Types of genes modulated by TgGCN5-A during alkaline stress.
Pie chart displays GO categories of genes that were not up-regulated in ΔGCN5-A parasites relative to wild-type (p<0.001) after culture in alkaline (pH 8.2) conditions.
Figure 5.
TgGCN5-A is enriched at genes up-regulated during alkaline pH stress.
Toxoplasma parasites stably expressing FLAG-tagged TgGCN5-A were maintained in HFFs with normal (pH 7.0) or alkaline (pH 8.2) medium prior to harvesting for qChIP using anti-FLAG antibody. Immunoprecipitated DNA was examined using primers to pH responsive genes phosphatidylinositol 3- and 4-kinase (PI3K), and protein kinase (PK) (A), or housekeeping gene controls, GAPDH and actin, ACT (B). Results are represented as a ratio of ChIP/Input DNA. The difference between pH 7 and pH 8 for PI3K is statistically significant (p = 0.02, Student's t-test).
Figure 6.
ΔGCN5-A parasites fail to up-regulate developmental genes in response to alkaline pH.
Wild-type (WT), ΔGCN5-A (KO), or complemented ΔGCN5-A (KO-C) parasites were incubated in control (pH 7.0) or alkaline media (pH 8.2) for four days. The levels of mRNA expression for stress-induced bradyzoite genes BAG1 (A) and LDH2 (B) or the constitutive gene actin, ACT1, (C) were monitored by quantitative real-time PCR. Error bars indicate the S.D. D. ChIP of FLAG-tagged TgGCN5-A performed as described in Fig. 5, but using primers for BAG1 and LDH2 (Table S6). Results are represented as a ratio of ChIP/Input DNA. P value for BAG1 sample = 0.02, P value for LDH2 sample = 0.01 (Student's t-test).
Figure 7.
Parasites lacking TgGCN5-A are defective in recovering from alkaline stress.
Equal numbers of extracellular wild-type (WT), ΔGCN5-A (KO), or complemented ΔGCN5-A (KO-C) parasites were subjected to alkaline (pH 8.2) or control (pH 7) media for 30 min at 37°C in 5% CO2. Following treatment, the parasites were allowed to infect HFF monolayers and incubated under normal culture conditions. A. Parasite growth was monitored with a standard plaque assay at day 5 (* denotes p = 0.017). B. Viability assay was set up as described above, but growth was monitored with the PCR-based B1 assay (** denotes p = 0.012).
Figure 8.
Response of ΔGCN5-A parasites to other stress conditions.
A. Equal numbers of extracellular wild-type (WT) or ΔGCN5-A (KO) were subjected to one of the following stresses for 30 min before being placed back into HFF culture under normal growth conditions: 0.6 M KCl, 5 µM sodium arsenite (NaAr), or 1 µM ionophore (Ion). Alkaline pH (8.2) stress was used as a positive control and no stress as a negative control. The mean number of plaques after five days recovery in HFF monolayer culture is shown. P values for Student's t-test are denoted as * = 0.03 and ** = 0.02. B. Equal numbers of extracellular wild-type (WT) or ΔGCN5-A (KO) were incubated at 37°C or 42°C for 30 minutes then placed in HFF culture at 37°C. The number of parasites in culture at day 5 post-infection was determined using the PCR-based B1 assay.