Trypanosoma cruzi dysregulates expression profile of piRNAs in primary human cardiac fibroblasts during early infection phase

Figures

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  Figure 1. RNA-seq analysis of small RNAs dysregulated by T. cruzi infection of PHCF. (A) Principle component analysis of PHCF gene expression was completed for all samples and probe sets. The x-axis is PC1 while the y-axis is PC2, with the percentage of variance indicated in parenthesis. PHCF was challenged with T. cruzi for various time points (0, 1, 3, and 6) denoted by circle and clustering (0 h = purple, 1 h = green, 3 h = red, 6 h = blue). (B) Heatmap and hierarchical clustering was performed on control and test samples 0, 1, 3, and 6 h, respectively, using Euclidean distance measure and single linkage analysis. Each column represents one sample and each row represents one small non-coding RNA within the data set. (C–E) Differential expressed putative and known piRNAs induced during parasite challenge of PHCF at 1, 3, and 6 hours respectively, belong to various transposable element subfamilies.

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  Figure 2. Dysregulation of piRNA expression in during early T. cruzi infection of PHCF. (A) Violin Plot of differentially expressed piRNA at each time point. The boxplot indicates the median, quartiles, and extremes of piRNA fold change. The width of the colored areas represents the proportion of piRNAs exhibiting a given level of expression. Volcano plots of piRNA expression at 1 (B), 3 (C), and 6 (D) hours post-T. cruzi challenge. The x-axis indicates the log2Fold change relative to the 0-hour control. The y-axis shows the q value. Upregulated and downregulated piRNAs are shown in red and blue, respectively. The criteria for statistically significant differential expression was log2Fold change >1, q >.8, and are indicated by the x-intercept and y-intercepts. The 5 selected piRNAs evaluated in our study are labeled in each plot. (E) Venn diagram of all upregulated (red) and downregulated (blue) piRNAs at various time points after T. cruzi challenge with FDR < 0.05. (F) Venn diagram of known piRNAs and (G) putative piRNAs dysregulated over the course of infection. These piRNAs previously not published have been deposited into NCBI SRA database.

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  Figure 3. Differentially expressed piRNAs dysregulated by T. cruzi infection map to specific genetic regions. The miRanda algorithm was used to predict potential target binding sites of both known and putative piRNAs. In these cartoons, exons represented by blue blocks, 5’ and 3’ untranslated regions (UTRs) are orange, and intronic regions are black. piRNAs predicted to target (A) ICAM1, (B) SMAD2, (C) CX3CL1, (D) CXCR2, and (E) EGR1.

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  Figure 4. Early T. cruzi infection dysregulates expression of piRNAs and their targets. (A–J). Total RNA and small RNA were purified from PHCF challenge with invasive T. cruzi trypomastigotes. Target transcript expression was evaluated using RT-qPCR, while piRNA expression was validated using stem-loop PCR for cDNA synthesis and RT-qPCR for relative expression. Bar graphs show log10 fold change ± SEM (n=3). Orange bar graphs denote relative mRNA target transcript expression while green bar graphs represent relative piRNA expression. Student’s t-test and visualization performed using GraphPad Prism. ns = not significant, *p < .05, **p < .01, ***p < 0.001, ****p < 0.0001.

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  Figure 5. Network of differentially expressed piRNAs targeting adhesion and chemokine signaling molecules operating during T. cruzi challenge of PHCF. Biological interaction networks were created using predicted piRNA targets (ICAM1, CX3CL1, and CXCR2) as primary seed nodes to query pathway and interaction data sources out to one degree of biological interaction. Primary seed notes are denoted as orange circles. Expansion notes (blue) with a predicted binding target of DE piRNAs (green).

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  Figure 6. Network of DE piRNAs targeting profibrotic transcription factors during PHCF challenged with T. cruzi. Biological interaction networks were created using predicted piRNA targets (SMAD2, FOS, and EGR1) as primary seed nodes to query pathway and interaction data sources out to one degree of biological interaction. Primary seed notes are displayed as orange circles. Expansion notes (blue) with a predicted binding target of DE piRNAs (green).