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. 2018 May 22;7:e36491. doi: 10.7554/eLife.36491

Figure 3. mRNAseq comparisons between T. gondii and H. hammondi identify unique aspects of the H. hammondi transcriptome.

(A) Venn diagrams indicating the degree of overlap in genes of significantly different transcript abundance between T. gondii and H. hammondi at D4 and D15 post-infection. While overall there are distinct genes that are significantly different at D4 and D15 (Venn diagram, upper left), H. hammondi expresses high numbers of bradyzoite genes even at 4 dPI and this number increases further by D15 (21 additional genes; Venn diagram in upper right). This progression towards an increase in the number of bradyzoite genes over time is not recapitulated among merozoite-specific genes, in that only four additional detectable genes from this gene set were found to be of significantly higher abundance (Padj <0.01; Fold-change ≥4) at 15 DPI. Moreover 18 of the 22 total detectable merozoite genes were of significantly different abundance in H. hammondi, indicating its transcriptional similarity to both bradyzoites and merozoites. (B) Gene Sets found to be significantly (FDR q-value <0.05) enriched in H. hammondi high-abundance transcripts (top) or T. gondii high-abundance transcripts (bottom) at either 4 or 15 days in culture. Gene Set Details can be found in Table 2. *: p<0.05; **: p<0.01; ***: p<0.001. (C) GSEA plots of the in vitro bradyzoite, in vitro tachyzoite, and Cat stage specific one gene sets, showing enrichment profiles between H. hammondi and T. gondii at 15 DPI. NES; Normalized enrichment score. FDR q: False discovery rate q-value. (D) Hierarchically clustered expression data for detectable genes in the CAT STAGE SPECIFIC one gene set in H. hammondi or T. gondii grown for 4 or 15 days in vitro, or for T. gondii grown as tachyzoites or merozoites (in cat enteroepithelial cells).

Figure 3.

Figure 3—figure supplement 1. Transcriptional profiling and Gene Set Enrichment Analysis of T. gondii and H. hammondi.

Figure 3—figure supplement 1.

(A) Summary of reads obtained for each parasite species and life stage, and the percentage of reads from each that mapped to the respective parasite genomes or transcriptomes. (B) Log2-transformed and normalized Fragments Per Million (FPM) data for the 4276 shared genes between T. gondii and H. hammondi that passed thresholds for detection in both species. Genes of significantly different abundance (based on criteria as listed in inset) are blue. (C) Hierarchical cluster (Euclidean distance, complete) of all detectable genes belonging to the IN VITRO TACHYZOITE gene set. Raw Log2 transformed FPM values (taken from DESeq2 following normalization and transformation using ‘rlog’) are shown. (D) Hierarchical cluster (Euclidean distance, complete) of all detectable genes belonging to the IN VITRO BRADYZOITE gene set. Raw Log2 transformed FPM values (taken from DESeq2 following normalization and transformation using ‘rlog’) are shown. Gene sets are described in Table 2.
Figure 3—figure supplement 2. mRNA-seq comparisons between TgVEG and HhCatAmer sporozoites identify unique H. hammondi transcriptome profiles.

Figure 3—figure supplement 2.

(A) A heat map illustrates differential expression profiles of cat stage specific genes enriched for TgVEG and HhCatAmer. The relative levels of gene expression (mean-centered log2-fold change) are depicted using a color scale where blue indicates the lowest level of expression and yellow indicates the highest level of expression. (B) Gene sets found to be enriched in H. hammondi high-abundance transcripts at 1 day post-excystation. Gene set details can be found in Table 2. (***: FDR q < 0.001; **: FDR q < 0.01). The AP2 transcription factor gene set is provided as a negative control. (C) GSEA plot of cat stage specific one gene set, showing enrichment profiles between H. hammondi and T. gondii at 1 day post-excystation. NES: Normalized enrichment score. FDR q: False discovery rate q-value. (D) A summary of reads obtained for each parasite species, and the percentage of reads from each that mapped to the respective parasite genomes or transcriptomes. A total of 8912 genes that had at least one read in both TgVEG and HhCatAmer samples were selected for analysis.
Figure 3—figure supplement 3. qPCR validation for nine transcripts that were found to be of higher abundance in D4 or D15 H. hammondi compared to T. gondii.

Figure 3—figure supplement 3.

(A) Clustered Log2(TPM) value taken from RNAseq expression profiles of HhCatEth1 and TgVEG at 4 and 15 DPI. Gene models are from TgME49 since that is the most well-annotated T. gondii genome and upon which our synteny maps were based. (B) qPCR for nine transcripts show significantly higher transcript level in HhCatEth1 compared to TgVEG at D4 pi. Fold difference of HhCatEth1 genes relative to TgVEG is shown; bars represent mean and SD from three biological replicates. Significance was determined from ΔCt values using multiple t-tests and the Holm-Sidak method, with alpha = 5.0%. Apical membrane antigen 1 (AMA1) and calcium dependent protein kinase 1 (CDPK1) genes served as examples of transcripts found to not differ between species and transcript abundance values were normalized using parasite GAPDH genes. All genes were queried using species-specific primers.