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Dual RNA-seq analysis of pig alveolar macrophage infected with china-isolated TgHB1 compared to TgRH and TgMe49 toxoplasma gondii standard strain

ABSTRACT: Toxoplasma gondii is an apicomplexan parasite infecting human and animals, causing huge health concerns and economic losses. However, it is unclear about the exact mechanism of T.gondii tachyzoite infected macrophage and macrophage resisted T.gondii, especially for local isolates such as TgHB1 isolated in China. Our study focused on the transcriptional difference of pig alveolar macrophages (3D4/21) infected with china isolated TgHB1 compared to TgRH and TgME49 toxoplasma gondii standard strains.

ORGANISM(S): Toxoplasma gondii Sus scrofa

PROVIDER: GSE153330 | GEO | 2020/09/12

REPOSITORIES: GEO

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Project description:Toxoplasma gondii is an obligate intracellular protozoan of severe threat to humans and livestock. Macrophages are the cell type preferentially infected by T. gondii in vivo. Protein phosphorylation is an important post-translational modification involved in diverse cellular functions. ARAF is member of the RAF family of serine/threonine protein kinases that is necessary for MAPK activation. Limited information is available about the level of protein phosphorylation variations and the roles of ARAF in macrophages with T. gondii infection, although our previous research found that ARAF knockout could reduce the apoptosis of porcine alveolar macrophages (3D4/21 cells) caused by T. gondii infection. Here, we used IMAC in combination with LC-MS/MS to profile the changes of phosphorylation in 3D4/21 cells and 3D4/21-ΔAraf cells upon Toxoplasma infection, respectively. A total of 1647 differentially expressed phosphorylated proteins (DEPPs) with 3876 differentially phosphorylated sites (DPSs) were identified in 3D4/21 cells with Toxoplasma infection (p3T group) when comparing 3D4/21 cells without parasite infection (pho3 group), and 959 DEPPs with1540 DPSs when comparing 3D4/21-ΔAraf cells with parasite infection (p3KT group). In addition, 552 DPSs corresponding to 406 DEPPs with the same phosphorylated sites were screened in the comparison p3T/pho3 vs. p3T/p3KT, which was identified as the DPSs and DEPPs related with ARAF. Remarkable functional properties of the DEPPs were discovered by GO analysis, KEGG pathway analysis, and STRING analysis. Of 406 DEPPs related with ARAF, 40 DEPPs corresponding to 57 DPSs involved in the apoptosis of 3D4/21 cells during Toxoplasma infection. Further analysis showed that the phosphorylation levels of MED1at serine1418, JUN at serine 73, MYC at serine 154, MCL1 at serine 65, and BAD at serine115 were upregulated in p3T, but downregulated in p3KT, suggesting that ARAF regulate phosphorylation of these sites to modulate the apoptosis of macrophages induced by Toxoplasma infection. These results revealed distinct responses of macrophages to Toxoplasma infection and the potential roles of ARAF in fighting against infection via phosphorylation of crucial proteins.

Project description:Intracellular pathogens including the apicomplexan and opportunistic parasite Toxoplasma gondii profoundly modify their host cells in order to establish infection. We have shown previously that intracellular T. gondii inhibit up-regulation of regulatory and effector functions in murine macrophages (MΦ) stimulated with interferon (IFN)-γ, which is the cytokine crucial for controlling the parasites’ replication. Using genome-wide transcriptome analysis we show herein that infection with T. gondii leads to global unresponsiveness of murine macrophages to IFN-γ. More than 61% and 89% of the transcripts, which were induced or repressed by IFN-γ in non-infected MΦ, respectively, were not altered after stimulation of T. gondii-infected cells with IFN-γ. These genes are involved in a variety of biological processes, which are mostly but not exclusively related to immune responses. Analyses of the underlying mechanisms revealed that IFN-γ-triggered nuclear translocation of STAT1 still occurred in Toxoplasma-infected MΦ. However, STAT1 bound aberrantly to oligonucleotides containing the IFN-γ-responsive gamma-activated site (GAS) consensus sequence. Conversely, IFN-γ did not induce formation of active GAS-STAT1 complexes in nuclear extracts from infected MΦ. Mass spectrometry of protein complexes bound to GAS oligonucleotides showed that T. gondii-infected MΦ are unable to recruit non-muscle actin to IFN-γ-responsive DNA sequences, which appeared to be independent of stimulation with IFN-γ and of STAT1 binding. IFN-γ-induced recruitment of BRG-1 and acetylation of core histones at the IFN-γ-regulated CIITA promoter IV, but not β-actin was diminished by >90% in Toxoplasma-infected MΦ as compared to non-infected control cells. Remarkably, treatment with histone deacetylase inhibitors restored the ability of infected macrophages to express the IFN-γ regulated genes H2-A/E and CIITA. Taken together, these results indicate that Toxoplasma-infected MΦ are unable to respond to IFN-γ due to disturbed chromatin remodelling, but can be rescued using histone deacetylase inhibitors. Comparison of 4 different RNA pools with a 2-Color-Loop Design including 10 microarrays: [1] T. gondii infected and IFN-gamma treated, [2] T. gondii infected and untreated, [3] Non-infected and IFN-gamma treated, and [4] Non-infected and untreated.

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Dual RNA-seq analysis of pig alveolar macrophage infected with china-isolated TgHB1 compared to TgRH and TgMe49 toxoplasma gondii standard strain

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