Phosphoproteomic analysis reveals changes in A-Raf-related protein phosphorylation in response to Toxoplasma gondii infection in porcine macrophages

Abstract

Background: Toxoplasma gondii is an obligate intracellular protozoan parasite that causes severe threats to humans and livestock. Macrophages are the cell type preferentially infected by T. gondii in vivo. Protein phosphorylation is an important posttranslational modification involved in diverse cellular functions. A rapidly accelerated fibrosarcoma kinase (A-Raf) is a member of the Raf family of serine/threonine protein kinases that is necessary for MAPK activation. Our previous research found that knockout of A-Raf could reduce T. gondii-induced apoptosis in porcine alveolar macrophages (3D4/21 cells). However, limited information is available on protein phosphorylation variations and the role of A-Raf in macrophages infected with T. gondii.

Methods: We used immobilized metal affinity chromatography (IMAC) in combination with liquid chromatography tandem mass spectrometry (LC-MS/MS) to profile changes in phosphorylation in T. gondii-infected 3D4/21 and 3D4/21-ΔAraf cells.

Results: A total of 1647 differentially expressed phosphorylated proteins (DEPPs) with 3876 differentially phosphorylated sites (DPSs) were identified in T. gondii-infected 3D4/21 cells (p3T group) when compared with uninfected 3D4/21 cells (pho3 group), and 959 DEPPs with 1540 DPSs were identified in the p3T group compared with infected 3D4/21-ΔAraf cells (p3KT group). Venn analysis revealed 552 DPSs corresponding to 406 DEPPs with the same phosphorylated sites when comparing p3T/pho3 versus p3T/p3KT, which were identified as DPSs and DEPPs that were directly or indirectly related to A-Raf.

Conclusions: Our results revealed distinct responses of macrophages to T. gondii infection and the potential roles of A-Raf in fighting infection via phosphorylation of crucial proteins.

Keywords: Toxoplasma gondii; 3D4/21; A-Raf; Apoptosis; Host cells; Phosphorylation.

Fig. 1

Identification and quantification of phosphopeptides. A Results for protein in the sample detected by SDS–PAGE (Coomassie brilliant blue staining). B Results for total threonine (Thr) phosphorylation levels detected by western blot; C Distribution of phosphorylation on serine (pSer), threonine (pThr), and tyrosine (pTyr) for all phosphorylation sites

Fig. 2

Analysis of differentially phosphorylated proteins. A The histogram of differentially expressed phosphorylated proteins (DEPPs). B, C Volcano plots of differentially phosphorylation sites. The horizontal axis is the log₂-transformed value of the differential expression change ratio of the comparison group; the vertical axis is the −log₁₀-transformed value of the statistical test t-test P value; where red points indicate significant upregulation of differential modifier loci (P < 0.05), blue points indicate significant downregulation (P < 0.05), and gray points indicate no significant difference (P > 0.05). The top five (absolute log₂ ratio from largest to smallest) differentially modified loci are also marked on the graph. D, E Cluster heat map of differentially phosphorylation sites. One differentially modified site per row, one sample per column. Red represents high expression, blue represents low expression. F Venn diagram showing the distribution of the differentially phosphorylated sites in two comparison groups

Fig. 3

Heat maps of enrichment of six amino acid motifs upstream and downstream of serine A and threonine B modification sites. All the identified differentially phosphorylated sites (DPSs) data were used for the analyses. The letters on the right of each panel represent amino acids. Red indicates that this amino acid is significantly enriched near the modification site, and green indicates that the secondary amino acid is significantly reduced near the modification site

Fig. 4

Functional enrichment of DEPPs of p3T/pho3. The X axis indicates the number of DEPPs. The Y-axis represents GO terms (A) and KEGG pathway maps (B). C The scatter plots represent KEGG pathway enrichment of the DEPPs of p3T/pho3. KEGG pathway analysis of DEPPs: the vertical axis shows the significantly enriched KEGG pathways and the horizontal axis represents the rich factors corresponding to the pathways. Rich factor refers to the ratio of the number of DEPPs to the number of all phosphoproteins in the pathway. Higher rich factors indicate greater degrees of enrichment. The size and color of the node represent number of phosphoproteins and P value of pathways

Fig. 5

Functional enrichment of DEPPs related to A-Raf and regulated during T. gondii infection. DEPPs data set used for the functional analyses was the DEPPs with the same phosphorylated sites in the comparison of p3T/pho3 versus p3T/p3KT. The X axis indicates the number of DEPPs. The Y axis represents GO terms (A) and KEGG pathway maps (B). C KEGG pathway enrichment of the DEPPs related to A-Raf

Fig. 6

Protein–protein interaction (PPI) networks of the DEPPs related to A-Raf. Cytoscape software and String database were used to construct the PPI networks. Nodes represent differentially expressed proteins. Size of the node represents number of the differential proteins and their interacting proteins

Fig. 7

KEGG pathway enrichment of the DEPPs related to A-Raf associated with apoptosis