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

## TL;DR

This study shows how protein phosphorylation changes in pig macrophages infected with Toxoplasma gondii, highlighting the role of A-Raf in the immune response.

## Contribution

The study identifies specific phosphorylation changes linked to A-Raf in T. gondii-infected macrophages using phosphoproteomic analysis.

## Key findings

- 1647 phosphorylated proteins with 3876 sites changed in infected cells compared to uninfected cells.
- 552 phosphorylation sites were shared between infected and A-Raf knockout cells, indicating A-Raf's role in infection response.
- A-Raf appears to influence macrophage responses to T. gondii through phosphorylation of key proteins.

## Abstract

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.

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.

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.

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.

The online version contains supplementary material available at 10.1186/s13071-024-06273-x.