- and MyD88. - Only a minimal number of transcripts were differentially expressed between MyD88 knockout and wild type cells. - gondii genes during infection, no differentially expressed parasite genes were identified when comparing infection in MyD88 knockout and wild type host BMDM.. - Dual RNA sequencing of Toxoplasma-infected macrophages identifies host and parasite transcripts We infected wild type and MyD88 knockout (KO) bone marrow-derived macrophages with both Type I (RH) and Type II (PTG) Toxoplasma tachyzoites, then col- lected samples 6 h later for high throughput RNA-seq. - gondii infection of wild type and MyD88 KO mouse macrophages. - Among the wild type mouse sam- ples, DE transcripts were primarily protein-coding (51%) and non-coding (43. - A complete listing of DE transcripts for the three wild type comparisons (RH vs uninfected, PTG vs uninfected, and RH vs PTG) is shown in Additional file 6.. - 88, MyD88 KO BMDM. - wt, wild type BMDM. - Many noncoding transcripts are differentially expressed during infection of wild type BMDM. - The majority (63%) of the differentially expressed noncoding tran- scripts identified in wild type BMDM are classified in GENCODE/Ensembl as retained_intron noncoding RNAs, defined as alternatively spliced transcripts be- lieved to contain intronic sequences relative to other coding transcripts of the same gene (Fig. - 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. - 4 Many non-coding transcripts are differentially expressed during infection with Toxoplasma. - a Classification of differentially expressed mouse non-coding transcripts by type. - 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. - Sequencing of MyD88 KO mouse Transcriptome reveals few gene expression differences between MyD88. - The breakdown of all DE transcripts by type was re- markably similar for infection of wild type and MyD88 KO mice (Fig. - In general, the overall number of protein-coding and noncoding DE transcripts for RH and PTG infection was similar for wild type and MyD88 KO BMDM (Figs. - Likewise, the number of DE transcripts shared between RH and PTG was similar for wild type and MyD88 KO macrophages (Figs. - A full list of the protein-coding and noncoding DE transcripts for MyD88 KO macrophages is provided in Additional file 6 and Additional file 10.. - Only a few protein-coding and noncoding tran- scripts were differentially expressed between wild type and MyD88 KO BMDM (Figs. - 10 protein- coding and 5 noncoding transcripts were differentially expressed between uninfected wild type and MyD88 KO macrophages. - Similarly, 9 protein-coding and 7 noncod- ing transcripts were differentially expressed between RH infected wild type and MyD88 KO macrophages. - 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. - a Classification of differentially expressed MyD88 KO transcripts as either protein-coding, non-coding, pseudogene, or TEC (To be Experimentally Confirmed). - c Venn diagrams of differentially expressed protein-coding transcripts. - d Classification of differentially expressed MyD88 KO non-coding transcripts by type. - 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. - d List of all differentially expressed protein-coding transcripts between wild type and MyD88 KO mice. - 10 protein-coding and 12 noncoding transcripts were differentially expressed between PTG infected MyD88. - In general, there was substantial overlap between the DE transcripts for wild type and MyD88 KO macro- phages ranging from ~ 20–50% depending on the infec- tion treatment (coding transcripts, Fig. - Functional analysis of the similar- ities between RH-infected wild type and MyD88 KO macrophages revealed enrichment of genes for cell cycle,. - macrophages, despite differ- ential expression of several immune genes such as ccl17, arg1, socs2, and ccl24 during RH infection of wild type and MyD88 KO cells. - Regarding lncRNAs, greater abundance of mir17hg was common to both wild type and MyD88 KO infection by RH and PTG.. - Siva1–205 and Nfkb1–210 lncRNAs were of higher abundance during RH infection of wild type and Myd88 KO macrophages.. - 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. - d List of all differentially expressed non- coding transcripts between wild type and MyD88 KO BMDM. - Transcripts encoding the macrophage receptor Marco and the cytokine Csf3 were of lower abundance in the MyD88 KO BMDM relative to wild type cells for both uninfected and PTG treatment (Fig. - Transcripts for Arg1 (encoding arginase that cata- lyzes arginine hydrolysis) were less abundant in unin- fected MyD88 KO macrophages. - Toxoplasma is an arginine auxotroph and upregulation of Arg1 in wild type cells is thought to be a host defense mechanism to reduce levels of arginine [35, 49]. - Ifi208 (interferon acti- vated gene 208) is more abundant in MyD88 KO BMDM compared to wild type during RH infection.. - wild type uninfected, MyD88 KO RH versus wild type RH, and MyD88 KO PTG versus wild type PTG) suggesting these two lncRNAs may be important in the difference be- tween the two macrophage strains (Fig. - One lncRNA, Sltm-207, is intronic to the protein-coding gene SLTM and is less abundant in MyD88 KO compared to wild type during PTG infection. - Sequencing of the Toxoplasma Transcriptome revealed no differences in gene expression between infection of wild type and MyD88 KO macrophages. - We directly compared RH and PTG infection for both wild type and MyD88 KO macrophages. - We also compared wild type versus MyD88 KO, for RH and PTG, respectively.. - For both wild type and MyD88 KO, approximately 120 parasite genes were more abundant in RH infection compared to PTG infection (Fig. - Surprisingly, no differen- tially expressed Toxoplasma genes were identified when comparing MyD88 KO RH versus wild type RH and MyD88 KO PTG versus wild type PTG host strains (Fig.. - Most (approximately 80%) of the differentially expressed genes between RH and PTG were shared between wild type and MyD88 KO strains (Fig. - gondii infection of wild type and MyD88 KO strains.. - However, two dynein proteins were more abundant in RH versus PTG for both wild type and MyD88 KO. - Many secretory proteins (ROP38, GRA11, ROP28, ROP2A, ROP23, ROP46, MIC17B, and ROP4) were less abundant in RH versus PTG in both wild type and MyD88 KO. - The rhoptry pro- tein ROP8 was less abundant in RH versus PTG only in MyD88 KO host cells. - Oocyt wall protein (OWP1) was less abundant in RH versus PTG in both wild type and MyD88. - Transcripts for the bradyzoite antigen BAG1 were less abundant during RH infection compared to PTG infection in both wild type and MyD88 KO. - Using normalized expression values, we com- pared the top 100 expressed genes between all four sam- ples (RH infection of wild type and MyD88. - PTG infection of wild type and MyD88. - gondii infection of wild type and MyD88 KO BMDMs. - While not the primary focus of our study, we identified many similarities to previous studies in the wild type mouse protein-coding response, including dys- regulation of immune and metabolic genes. - genes were differentially expressed during Type I rather than Type II infection. - 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. - b Venn diagrams showing shared expression changes between wild type and MyD88 KO samples for RH vs. - 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. - Here, we found 249 noncoding transcripts as differentially expressed during Type I in- fection and 83 noncoding transcripts as differentially expressed during Type II infection of wild type mouse macrophages. - Add- itionally, 31 noncoding transcripts were differentially expressed in both RH and PTG infection. - Perhaps surprisingly, only a few genes were differen- tially expressed between wild type and MyD88 KO mice.. - Fur- ther investigation of these protein-coding genes and non- coding transcripts in the context of the MyD88 KO im- mune response is important.. - While not the primary focus of our study, we also ex- amined parasite gene expression for RH and PTG infec- tion of wild type and MyD88 KO macrophages. - than 550 genes were differentially expressed between Type I and Type II T. - Several microtubule and motor proteins (myosin, dynein, intraflagellar transport protein, and kinesin) were less abundant in RH versus PTG in both wild type and MyD88 KO. - gondii genes were differentially expressed between wild type and MyD88 KO macrophages, suggesting that T. - Nevertheless, it is possible that at later infection time points differences in the parasite transcriptome in wild type and KO cells would emerge.. - gondii infection and (2) examin- ing the transcriptional response of MyD88 KO cells. - C57BL/6 mice (The Jackson Laboratory) were used as the wild type strain in this study. - The MyD88 KO mice contain a deletion of exon 3 (The Jackson Laboratory,. - We used a total of 8 wild type mice and 8 MyD88 KO mice in this study. - Wild type Toxoplasma strains RH (Type I) and PTG (Type II) were used in this study. - Principal component analysis (PCA) plots of mouse RNA-sequencing data reveal associations between samples: PCA plots of mouse transcripts for MyD88 KO BMDM versus wild type BMDM comparisons.. - gondii transcripts for MyD88 KO (k88) BMDM versus wild type (wt) BMDM comparisons.. - Functional analysis of differentially expressed protein- coding genes. - Complete list of differentially expressed mouse non- coding genes. - Additional file 12 Complete list of differentially expressed Toxoplasma gondii genes. - The first tab corresponds to wild type RH versus wild type PTG. - DE: Differentially expressed
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