Figure 1.
Patterns of gene expression vary more by strain than by developmental stage.
A) A representative strain from T. gondii lineages Types I (RH), II (PLK), and III (CTG) was selected and grown in tissue culture in either pH neutral conditions, conducive to tachyzoite growth or alkaline conditions, inducing bradyzoite differentiation. B) Multi-dimensional scaling (MDS) plot based on pairwise comparisons for each of the six experimental conditions. This is calculated as the root mean square deviation for the 500 most differentially expressed genes between any two conditions. The distance between any two points represents the average difference in expression levels (RPKM) of the most dissimilar genes, relative to differences observed between other conditions. In effect, the MDS plot provides an overview of the total amount of variation between samples. The axes show arbitrary distances. Experimental conditions include parasite strain (red = RH, green = PLK, blue = CTG) and by life cycle stage (X = tachyzoite, O = bradyzoite). Distances calculated using the 'plotMDS' function in the 'limma' Bioconductor package [30]. C) Boxplot represents absolute value of expression level difference between conditions. Interquartile regions are in gold, median differences are plotted as a solid black line. The root mean square deviation for each comparison is represented as a blue cross. From left to right, the first three groups are the intrastrain comparisons, tachyzoite vs. bradyzoite. The next three groups are interstrain comparisons between tachyzoite (unstressed) groups. The final three are are interstrain comparisons between bradyzoite (alkaline-stressed) groups).
Figure 2.
Hundreds of genes differentially expressed between strains including potential AP2 regulators.
A) Using edgeR, we identify genes that are differentially expressed between strains (FDR <10%), generating three gene lists for tachyzoite condition and three for the bradyzoite condition. We generated Venn diagrams showing the overlap between the lists in the tachyzoite condition and bradyzoite condition. We also compared tachyzoite and bradyzoite gene lists to each other. Venn diagrams shows comparison of genes that appear in both the tachyzoite and the bradyzoite lists and therefore are “stage independent”. AP2 containing genes appearing on more than one list (any intersection) are indicated. The complete set of genes that are differentially expressed are listed in Table S2 and RPKM values for all replicates are listed in Table S1. B) Heat map of genes differentially expressed following CTG bradyzoite differentiation in all six conditions. Red indicates up regulation compared to that gene's expression level under other conditions, whereas green indicates down regulation. Conditions (columns) are clustered based on similarity of expression levels. The five AP2 genes that are differentially expressed are indicated on the right. Heat map was generated using the 'heatmap.2' function in the 'gplots' package for R. Hierarchical clustering of both the rows (genes) and columns (conditions) computed by the 'hclust' function in the R 'stats' package. Based on mean of replicate RPKM values.
Figure 3.
GSEA detects bradyzoite-induced genes in PLK and CTG, but not RH parasites.
(A) A schematic of strain virulence of the strains used as a function of ability to differentiate into bradyzoites. (B) GSEA enrichment plot for RH parasites under differentiation conditions compared to compound1 induced genes. Position of black bars indicate ranking of compound 1 genes relative to all other genes. Green line represents strength of enrichment under bradyzoite conditions (right) or tachyzoite conditions (left). (C) Enrichment plot for PLK parasites under differentiation conditions compared to compound 1 induced genes. (D) Enrichment plot for CTG parasites under differentiation conditions compared to compound 1 induced genes.
Table 1.
Differential expression of KEGG pathways between strains in the tachyzoite stage.
Table 2.
Differential expression of KEGG pathways between strains in treated with alkaline stress bradyzoite induction conditions.
Figure 4.
Expression of cell-cycle genes altered during differentiation.
A) Boxplot represents differences in expression levels of cell cycle-dependent genes following differentiation conditions. Green boxes represent changes in S/M gene expression, blue boxes represent changes in G1 gene expression. A positive difference indicates up regulation of the gene in tachyzoite conditions, while a negative difference in expression values indicates greater expression in the alkaline stress induced bradyzoites. The black bar indicates median value; the red cross indicates mean value. Significance tested by one-way ANOVA. A star (*) indicates: p<0.001. B) Cell cycle genes are annotated as either G1 or S/M [8]. We then sorted genes into groups based on time of peak expression. Each of these gene sets was tested by GSEA [26]. Gene sets with significant (FWER-p value <0.05) normalized enrichment scores (NES) are plotted. Positive scores indicate association with the unstressed (tachyzoite) condition, negative scores indicate association with the stress (bradyzoite) condition. Blue and green bar across middle of plots represent an eight hour RH tachyzoite cell cycle. Counter-clockwise from the top, the plots show cell cycle gene sets influenced following RH differentiation, cell cycle gene sets influenced following PLK differentiation, cell cycle gene sets influenced following CTG differentiation. Note that time of expression is based on the RH cell cycle as defined, which is shorter than that of either PLK or CTG.