Trypanosoma cruzi Modulates PIWI-Interacting RNA Expression in Primary Human Cardiac Myocytes during the Early Phase of Infection

Figures

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  Figure 1. Analysis of RNA-seq data and putative piRNAs induced by T. cruzi mapped to different transposable element subfamilies. (A) Principal component analysis (PCA) of gene expression was performed for all samples and all probe sets, by using a median centering of the data set. The x-axis corresponds to principal component 1 (PC1) and the y-axis to the principal component 2 (PC2); the percentage of the variance is indicated between brackets. Based on their small non-coding RNA expression values, samples from each time point (0, 1 and 2 h) clustered together, confirming homogeneity of the gene expression profiles within each group. The group of T. cruzi challenged samples clustered independently of the control group (blue = 0 h, yellow = 1 h, red = 2 h). (B) Heatmap and hierarchical clustering was performed on control and test samples, 0, 1 and 2 h, respectively, using Euclidean distance measure and single linkage analysis. Each column represents one sample and each row represents one non-small coding RNA within the data set. The color-coded scale illustrating the relative expression after global normalization is indicated. Differentially expressed putative and known piRNAs induced post…

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  Figure 2. T. cruzi induces differential expression of piRNAs in PHCM. (A) Venn diagram of upregulated and downregulated piRNAs counts at different time points after T. cruzi challenge of PHCM, FDR corrected p < 0.05. (B) Venn diagram generated showing the number of differentially expressed known piRNAs at 1 and 2 h post parasite challenge of PHCM. These previously reported and annotated piRNAs are available in the NCBI database. (C) Venn diagram showing distribution of putative piRNAs that were differentially expressed in PHCM at different time points after T. cruzi challenge. These piRNAs which were not previously published have now been deposited in the NCBI database.

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  Figure 3. piRNAs differentially expressed in PHCM during the early phase of T. cruzi infection mapped to specific genetic regions. miRANDA and RNA22 algorithms were used to predict target binding sites of known and novel piRNAs. In the genetic cartoons, exons represented by blocks are connected by horizontal lines representing introns while the 5′ and 3′ untranslated regions (UTRs) are represented as thinner blocks at the extremities of each gene, respectively. NFATC2 gene cartoons (3′ to 5′ orientation) showing positions where (A) known piRNAs and (B) novel piRNAs that are differentially expressed at 1, 2 h or both time points are predicted to be mapped, respectively. Cartoons of FOS gene (5′–3′) showing positions where (C) known piRNAs and (D) novel piRNAs that are differentially expressed at 1, 2 h or both time points are predicted to be mapped, respectively. (E) Cartoon of TGF-β1 gene (3′–5′) showing positions where differentially expressed novel piRNAs are mapped.

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  Figure 4. Networks of differentially expressed piRNAs targeting FOS and NFATC2 operating during PHCM challenge with T. cruzi. Biological interaction networks were created using predicted piRNA target genes (FOS, NFATC2) as primary seed nodes to query pathway and interaction data sources out to one degree of interaction. Primary seed nodes are displayed as red circles. Expansion nodes (blue circles) with a predicted binding target of a differentially expressed known or novel piRNA (yellow node) were added to the network. Connections between nodes (edges) represent interactions between the different biological entities. Networks of (A) known piRNAs and (B) novel piRNAs targeting FOS, NFATC2 and adjacent genes from expansion to one degree of interaction during all time points.

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  Figure 5. Network of differentially expressed novel piRNAs targeting TGF-β1 and heatmap of differentially expressed piRNAs in PHCM induced early during T. cruzi infection. Intensity plot of differentially expressed piRNAs, having greater than two-fold up- or downregulation (log2 ratio) with a probability of differential expression q-value ≥ 0.8. Color-coded scale illustrates the normalized relative expression. (A) Network of novel piRNAs targeting TGF-β1 (seed node) and adjacent genes to one degree of interaction at all time points. (B) Heatmap of differentially expressed known and novel piRNAs targeting NFATC2, FOS and TGF-β1 during the early infection phase.