Transcriptomics analysis of Toxoplasma gondii-infected mouse macrophages reveals coding and noncoding signatures in the presence and absence of MyD88

Abstract

Background: Toxoplasma gondii is a globally distributed protozoan parasite that establishes life-long asymptomatic infection in humans, often emerging as a life-threatening opportunistic pathogen during immunodeficiency. As an intracellular microbe, Toxoplasma establishes an intimate relationship with its host cell from the outset of infection. Macrophages are targets of infection and they are important in early innate immunity and possibly parasite dissemination throughout the host. Here, we employ an RNA-sequencing approach to identify host and parasite transcriptional responses during infection of mouse bone marrow-derived macrophages (BMDM). We incorporated into our analysis infection with the high virulence Type I RH strain and the low virulence Type II strain PTG. Because the well-known TLR-MyD88 signaling axis is likely of less importance in humans, we examined transcriptional responses in both MyD88^+/+ and MyD88^-/- BMDM. Long noncoding (lnc) RNA molecules are emerging as key regulators in infection and immunity, and were, therefore, included in our analysis.

Results: We found significantly more host genes were differentially expressed in response to the highly virulent RH strain rather than with the less virulent PTG strain (335 versus 74 protein coding genes for RH and PTG, respectively). Enriched in these protein coding genes were subsets associated with the immune response as well as cell adhesion and migration. We identified 249 and 83 non-coding RNAs as differentially expressed during infection with RH and PTG strains, respectively. Although the majority of these are of unknown function, one conserved lncRNA termed mir17hg encodes the mir17 microRNA gene cluster that has been implicated in down-regulating host cell apoptosis during T. gondii infection. Only a minimal number of transcripts were differentially expressed between MyD88 knockout and wild type cells. However, several immune genes were among the differences. While transcripts for parasite secretory proteins were amongst the most highly expressed T. gondii genes during infection, no differentially expressed parasite genes were identified when comparing infection in MyD88 knockout and wild type host BMDM.

Conclusions: The large dataset presented here lays the groundwork for continued studies on both the MyD88-independent immune response and the function of lncRNAs during Toxoplasma gondii infection.

Keywords: Macrophages; MyD88; Noncoding RNA; Parasite; Toxoplasma gondii; lncRNA.

Fig. 1

Flow chart demonstrating the steps taken to identify differentially expressed transcripts during T. gondii infection of wild type and MyD88 KO mouse macrophages

Fig. 2

Overview of RNA-sequencing reads mapping to both mouse and Toxoplasma genomes. A total of 20 RNA samples were submitted for sequencing, and characteristics of each sample are provided here. Column 1 denotes the sample name. 88, MyD88 KO BMDM; wt, wild type BMDM; M, noninfected macrophages; RH, macrophages infected with Type I RH strain Toxoplasma; PTG, samples infected with T. gondii Type II PTG strain. The numbers indicate independent biological replicates. InputReads (Column 2) denotes the number of reads obtained for each sample. Dropped % (Column 3) indicates the percent of input reads deemed low-quality and dropped. Mouse % (Column 4) is the percent of high-quality reads that mapped to the mouse genome. Toxo % (Column 5) denotes the percent of high-quality reads that mapped to the T. gondii genome. Shared % (Column 6) indicates the percent of high-quality reads mapping to both mouse and T. gondii genomes

Fig. 3

A much greater number of protein-coding genes are differentially expressed during infection with the highly virulent Toxoplasma RH strain than with the less virulent PTG strain. Wild type BMDM were infected with either the highly virulent RH strain or the less-virulent PTG strain, and 6 h later RNA was isolated for sequencing. Differentially expressed mouse transcripts were identified based on statistical significance (PPDE greater than 0.95) and a fold change of greater or less than 2. a Classification of differentially expressed mouse transcripts as either protein-coding, non-coding, pseudogene, or TEC (To be Experimentally Confirmed). b Total number of protein-coding transcripts of higher or lower abundance during infection. c Venn diagrams of differentially expressed protein-coding transcripts showing shared and unique expression patterns between infection strains. d Heat maps displaying trends among functionally related genes. Experiments were performed in at least triplicate with BMDM preparations from separate mice

Fig. 4

Many non-coding transcripts are differentially expressed during infection with Toxoplasma. Wild type BMDM were infected with either the highly virulent RH strain or the less-virulent PTG strain, and 6 h later RNA was isolated for sequencing. Differentially expressed mouse non-coding transcripts were identified based on statistical significance (PPDE greater than 0.95) and a fold change of greater or less than 2. a Classification of differentially expressed mouse non-coding transcripts by type. b Total number of non-coding transcripts of higher or lower abundance during infection. c Venn diagrams of differentially expressed non-coding transcripts revealing shared and unique expression patterns between infection strains. d List of all noncoding transcripts differentially expressed between RH and PTG. e List of all noncoding differentially expressed transcripts shared between RH and PTG infection. Experiments were performed in at least triplicate with BMDM from separate mice

Fig. 5

MyD88 KO BMDM have similar gene expression signatures as wild type BMDM. BMDM from MyD88 KO mice were infected with either the RH or PTG strain, and 6 h later RNA was isolated for sequencing. Differentially expressed mouse transcripts were identified based on statistical significance (PPDE greater than 0.95) and a fold change of greater or less than 2. a Classification of differentially expressed MyD88 KO transcripts as either protein-coding, non-coding, pseudogene, or TEC (To be Experimentally Confirmed). b Total number of protein-coding transcripts of higher or lower abundance during infection. c Venn diagrams of differentially expressed protein-coding transcripts. d Classification of differentially expressed MyD88 KO non-coding transcripts by type. e Total number of non-coding transcripts of higher or lower abundance during infection. f Venn diagrams of regulated non-coding transcripts revealing shared and unique expression patterns between infection strains. Experiments were performed in at least triplicate with BMDM from separate mice

Fig. 6

A minimal number of protein-coding genes were differentially expressed between wild type and MyD88 KO BMDM. Macrophages from wild type and MyD88 KO mice were infected with either the RH or PTG strain, and 6 h later RNA was isolated for sequencing. Differentially expressed mouse protein-coding transcripts between wild type and MyD88 KO BMDM were identified based on statistical significance (PPDE greater than 0.95) and a fold change of greater or less than 2. a Total number of protein-coding transcripts of higher or lower abundance between wild type and MyD88 KO mice for uninfected, RH-infected, and PTG-infected samples. b Venn diagrams of the gene expression differences between wild type and MyD88 KO mice showing shared expression changes for uninfected, RH-infected, and PTG-infected samples. c Venn diagrams revealing similarities between wild type and MyD88 KO gene expression changes for RH vs. uninfected, PTG vs. uninfected, and PTG vs. RH. d List of all differentially expressed protein-coding transcripts between wild type and MyD88 KO mice. Coloring indicates those transcripts shared between comparisons

Fig. 7

Only a few non-coding genes were differentially expressed between wild type and MyD88 KO BMDM. Cells from wild type and MyD88 KO mice were infected with Toxoplasma tachyzoites, and RNA was isolated for sequencing 6 h later. Differentially expressed mouse non-coding transcripts between wild type and MyD88 KO BMDM were identified based on statistical significance (PPDE greater than 0.95) and a fold change of greater or less than 2. a Total number of non-coding transcripts of higher or lower abundance between wild type and MyD88 KO macrophages for uninfected, RH-infected, and PTG-infected samples. b Venn diagrams of the non-coding gene expression differences between wild type and MyD88 KO cells revealing shared expression changes for uninfected, RH-infected, and PTG-infected samples. c Venn diagrams indicating similarities between wild type and MyD88 KO non-coding gene expression changes for RH vs. uninfected, PTG vs. uninfected, and PTG vs. RH. d List of all differentially expressed non-coding transcripts between wild type and MyD88 KO BMDM. Colored transcripts indicate those shared by different comparisons

Fig. 8

Greater than 550 Toxoplasma gondii genes were differentially expressed between the highly virulent RH strain and the less virulent PTG strain, but none were significantly different between wild type mice and MyD88 KO mice. BMDM from wild type and MyD88 KO mice were infected with RH or PTG tachyzoites, and 6 h later RNA was isolated for sequencing. Differentially expressed T. gondii genes were identified based on statistical significance (PPDE greater than 0.95) and a fold change of greater or less than 2. a Total number of T. gondii genes of higher or lower abundance for each comparison. b Venn diagrams showing shared expression changes between wild type and MyD88 KO samples for RH vs. PTG. c Pathways enriched between RH and PTG infection of wild type macrophages d Pathways enriched between RH and PTG infection of MyD88 KO BMDM. e Biological process enrichment between RH and PTG infection of wild type macrophages. f Biological process enrichment between RH and PTG infection of MyD88 KO cells

Fig. 9

Rhoptry, microneme, and dense granule genes are among the most abundant Toxoplasma transcripts. Using normalized expression values, the top 100 expressed genes were compared between the four samples (RH infecting wild type, RH infecting MyD88 KO, PTG infecting wild type, and PTG infecting MyD88 KO). 75 out of 100 were shared between all four samples and those shared are listed here