Phosphoproteomic analysis reveals the role of A-Raf in regulating the apoptosis of porcine macrophages infected with Toxoplasma gondii

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Abstract 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. A-Raf is member of the Raf family of serine/threonine protein kinases that is necessary for MAPK activation. Our previous research found that A-Raf knockout could reduce the apoptosis of porcine alveolar macrophages (3D4/21 cells) caused by T. gondii infection. However, limited information is available about the level of protein phosphorylation variations and the roles of A-Raf in macrophages with 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 obtained in the comparison p3T/pho3 vs. p3T/p3KT, which was identified as the DPSs and DEPPs related with A-Raf. Remarkable functional properties of the DEPPs were discovered by GO analysis, KEGG pathway analysis, and STRING analysis. Of 406 DEPPs related with A-Raf, 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 A-Raf 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 A-Raf in fighting against infection via phosphorylation of crucial proteins.

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