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.

中文翻译：

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磷酸化蛋白质组学分析揭示了猪巨噬细胞中弓形虫感染引起的 A-Raf 相关蛋白磷酸化的变化

弓形虫是一种专性细胞内原生动物寄生虫，对人类和牲畜造成严重威胁。巨噬细胞是体内弓形虫优先感染的细胞类型。蛋白质磷酸化是涉及多种细胞功能的重要翻译后修饰。快速加速纤维肉瘤激酶 (A-Raf) 是丝氨酸/苏氨酸蛋白激酶 Raf 家族的成员，是 MAPK 激活所必需的。我们之前的研究发现，敲除A-Raf可以减少弓形虫诱导的猪肺泡巨噬细胞（3D4/21细胞）的细胞凋亡。然而，关于蛋白质磷酸化变异和 A-Raf 在弓形虫感染的巨噬细胞中的作用的信息有限。我们使用固定金属亲和色谱 (IMAC) 结合液相色谱串联质谱 (LC-MS/MS) 来分析弓形虫感染的 3D4/21 和 3D4/21-ΔAraf 细胞中磷酸化的变化。与未感染的 3D4/21 细胞（pho3 组）相比，弓形虫感染的 3D4/21 细胞（p3T 组）中总共鉴定出 1647 个差异表达磷酸化蛋白（DEPP）和 3876 个差异磷酸化位点（DPS），并且检测到 959 个差异表达磷酸化蛋白（DEPP）。与感染的 3D4/21-ΔAraf 细胞（p3KT 组）相比，p3T 组中鉴定出具有 1540 DPS 的 DEPP。维恩分析发现，当比较p3T/pho3与p3T/p3KT时，552个DPS对应于406个具有相同磷酸化位点的DEPP，这被鉴定为与A-Raf直接或间接相关的DPS和DEPP。我们的结果揭示了巨噬细胞对弓形虫感染的独特反应以及 A-Raf 通过关键蛋白质磷酸化对抗感染的潜在作用。