Dual transcriptional profiling of mice and Toxoplasma gondii during acute and chronic infection

Abstract

Background: The obligate intracellular parasite Toxoplasma gondii establishes a life-long chronic infection within any warm-blooded host. After ingestion of an encysted parasite, T. gondii disseminates throughout the body as a rapidly replicating form during acute infection. Over time and after stimulation of the host immune response, T. gondii differentiates into a slow growing, cyst form that is the hallmark of chronic infection. Global transcriptome analysis of both host and parasite during the establishment of chronic T. gondii infection has not yet been performed. Here, we conducted a dual RNA-seq analysis of T. gondii and its rodent host to better understand host and parasite responses during acute and chronic infection.

Results: We obtained nearly one billion paired-end RNA sequences from the forebrains of uninfected, acutely and chronically infected mice, then aligned them to the genomic reference files of both T. gondii and Mus musculus. Gene ontology (GO) analysis of the 100 most highly expressed T. gondii genes showed less than half were shared between acute and chronic infection. The majority of the highly expressed genes common in both acute and chronic infection were involved in transcription and translation, underscoring that parasites in both stages are actively synthesizing proteins. Similarly, most of the T. gondii genes highly expressed during chronic infection were involved in metabolic processes, again highlighting the activity of the cyst stage at 28 days post-infection. Comparative analyses of host genes using uninfected forebrain revealed over twice as many immune regulatory genes were more abundant during chronic infection compared to acute. This demonstrates the influence of parasite development on host gene transcription as well as the influence of the host environment on parasite gene transcription.

Conclusions: RNA-seq is a valuable tool to simultaneously analyze host and microbe transcriptomes. Our data shows that T. gondii is metabolically active and synthesizing proteins at 28 days post-infection and that a distinct subset of host genes associated with the immune response are more abundant specifically during chronic infection. These data suggest host and pathogen interplay is still present during chronic infection and provides novel T. gondii targets for future drug and vaccine development.

Figure 1

Schematic of T. gondii /host dataset generation. (A) Nine mice were divided into three experimental groups: uninfected, 10 days post-infection, and 28 days post-infection. Infected mice were given 10,000 type II ME49 parasites and sacrificed on the corresponding days. The forebrains were removed and homogenized in TRIzol, and RNA was extracted and purified. A cDNA library was generated from the RNA prior to IlluminaHiSeq2000 sequencing. Raw reads were aligned to either the T. gondii or M. musculus genomes, normalized and analyzed for differential gene expression. (B) To examine T. gondii in the brains of mice at the designated time points, mice were infected with 10,000 parasites of a bioluminescent T. gondii[62]. Shown are representative brains for 10 and 28 day post-infection mice, after the brains were soaked for 5 minutes in luciferin prior to imaging.

Figure 2

Top 100  T. gondii genes from acute and chronic infection. (A) Venn diagram compares the 100 T. gondii genes with the greatest FPKM value from acute and chronic time points. Of the top 100 genes for each stage, 42 genes were similarly abundant (green), and 58 were only abundant in acute (blue) or chronic (yellow) infection. (B) GO terms were assigned to the top 100 T. gondii genes from acute and chronic infection. Genes were grouped based on whether they are similar or different between time points prior to GO term analysis.

Figure 3

More host genes have increased abundance during chronic infection. (A) DEGs in the mouse with a fold change >2 were grouped based on increased abundance (grey) and decreased abundance (black) between acute vs uninfected, chronic vs uninfected, and chronic vs acute time points. (B) A Venn diagram was created to compare DEGs with increased abundance in acute vs uninfected (purple) and chronic vs uninfected time points (red). Of the 1004 more abundant DEGs in acute vs uninfected, 902 were also more abundant in the chronic vs uninfected group (magenta). (C) To explore the function of DEGs analyzed in the Venn diagram, a GO term enrichment analysis was performed.

Figure 4

Analysis of host genes with decreased abundance during T. gondii infection. (A) A Venn diagram was created to compare DEGs >2-fold decreased abundance in acute vs uninfected (purple) and chronic vs uninfected (red) mice. Forty host genes were less abundant during both acute and chronic infection (magenta). (B) To explore the function of DEGs analyzed in the Venn diagram, a GO term enrichment analysis was performed. Functionally enriched GO terms display little similarity between time points.

Figure 5

Few highly expressed DEGs are specific to acute infection. (A) To better characterize abundant DEGs, a Venn diagram of the more abundant DEGs in the mouse with a fold change >20 between acute vs uninfected (purple) and chronic vs uninfected (red) time points. Of these more abundant DEGs, 146 were shared between acute and chronic infection (magenta). Only 9 DEGs had a fold change >20 in acute vs uninfected that were also not highly abundant in chronic vs uninfected time points. (B) To analyze the function of the host more abundant DEGs in chronic vs acute time points, GO term enrichment analysis was performed. Many genes differentially expressed in chronic infection are associated with immune regulation and stress response.