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Phosphoproteomic analysis reveals the role of ARAF in regulating the apoptosis of porcine macrophages infected with Toxoplasma gondii

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. 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.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Sus Scrofa Domesticus (domestic Pig)

TISSUE(S): Cell Culture, Macrophage

SUBMITTER: su dingzeyang

LAB HEAD: Su Dingzeyang

PROVIDER: PXD044725 | Pride | 2024-04-27

REPOSITORIES: Pride

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Project description:The innate immune response of mucosal epithelial cells during pathogen invasion plays a central role in immune regulation in the gut. Toxoplasma gondii (T. gondii) is a protozoan intracellular parasite that is usually transmitted through oral infection. Although much of the information on immunity to T. gondii has come from intra-peritoneal infection models, more recent studies have revealed the importance of studying immunity following infection through the natural per-oral route. Oral infection studies have identified many of the key players in the intestinal response; however, they have relied on responses detected days to weeks following infection. Much less is known about how the gut epithelial layer senses and reacts during initial contact with the pathogen. Given the importance of epithelial cells during pathogen invasion, this study uses an in vitro approach to isolate the key players and examine the early response of intestinal epithelial cells during infection by T. gondii. We show that human intestinal epithelial cells infected with T. gondii elicit rapid MAPK phosphorylation, NF-ÎºB nuclear translocation, and secretion of interleukin (IL)-8. Both ERK1/2 activation and IL-8 secretion responses were shown to be MyD88 dependent and TLR2 was identified to be involved in the recognition of the parasite regardless of the parasite genotype. Furthermore, we were able to identify additional T. gondii-regulated genes in the infected cells using a pathway-focused array. Together, our findings suggest that intestinal epithelial cells were able to recognize T. gondii during infection, and the outcome is important for modulating intestinal immune responses. Oral infection studies have demonstrated an increase in several cytokines and chemokines in response to T.gondii infection; however, the mixed population of the intestinal mucosa did not allow for the determination of the relative role that specific cell populations play in the production of these mediators. To address the role of intestinal epithelial cells to modulate the cytokine environment early following infection, we used specific pathway arrays to identify cytokines and chemokines induced 4 hours after exposure to T. gondii. At this time point most cells have become infected, but the parasites have not replicated.

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Phosphoproteomic analysis reveals the role of ARAF in regulating the apoptosis of porcine macrophages infected with Toxoplasma gondii

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