弓形虫钙依赖性蛋白激酶3通过靶向宿主精氨酸酶-1介导M1巨噬细胞极化。

Toxoplasma calcium-dependent protein kinases 3 mediates M1 macrophage polarization by targeting host Arginase-1.

作者信息

An Ran, Liu Fang, Dai Niuniu, Li Fangmin, Liu Xingyun, Cai Haijian, Chen Lijian, Du Jian

机构信息

Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Anhui Medical University, Hefei, 230032, China.

The Research Center for Infectious Diseases, School of Basic Medical Sciences, Anhui Medical University, Hefei, 230032, China.

出版信息

Parasit Vectors. 2025 May 20;18(1):181. doi: 10.1186/s13071-025-06799-8.

DOI:10.1186/s13071-025-06799-8

PMID:40394721

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC12090600/

Abstract

BACKGROUND

Toxoplasma gondii, an obligate intracellular parasite, has developed sophisticated ways to manipulate host immunity, resulting in long-lasting infection and causing serious public health problems in humans and animals. T. gondii type II is the type most frequently associated with human diseases, but the mechanism remains unclear. Toxoplasma calcium-dependent protein kinase 3(CDPK3), a protein located on the T. gondii parasite periphery, is highly expressed in type II strains. Although TgCDPK3 regulates parasite egress from host cells, calcium-based infiltration, and development of tissue cysts, the host target proteins that it modulates are still unclear.

METHODS

Firstly, mass spectrometry was used to analyze proteins that selectively bind to TgCDPK3. Subsequently, GST (glutathione-s-transferase) pull-down, immunoprecipitation, and immunofluorescence assay were used to confirm the interaction and colocalization between TgCDPK3 and Arginase-1. Western blotting and Argininaseactivity assays were performed to detect the relative levels of endogenous Arginase-1 and inducible nitric oxide synthase (iNOS) in a murine microglial cell line. Fluorescence activated cell sorting (FACS) assays and enzyme-linked immunosorbent assay (ELISA) analysis were performed to confirm the association of interaction between TgCDPK3 and Arginase-1 within an M1/M2-polarized macrophage. Intracellular multiplication assays and plaque assays were performed to test whether the interaction between TgCDPK3 and Arginase-1 affected intercellular parasite growth.

RESULTS

The interaction between TgCDPK3 and Arginase-1 is functionally correlated and leads to a reduction in Arginase-1 activity, ultimately, contributing to the M1-biased phenotype of the host macrophages, which is related to restraining the proliferation of parasites.

CONCLUSIONS

Our data showed that CDPK3 mediates M1 macrophage polarization by targeting host Arginase-1, which is beneficial to understanding the mechanism for long term latency establishment of less virulent strains of Toxoplasma.

摘要

背景

刚地弓形虫是一种专性细胞内寄生虫，它已形成复杂的方式来操纵宿主免疫，导致持续感染，并在人类和动物中引发严重的公共卫生问题。刚地弓形虫II型是最常与人类疾病相关的类型，但其机制仍不清楚。弓形虫钙依赖性蛋白激酶3（CDPK3）是一种位于刚地弓形虫寄生虫外周的蛋白质，在II型菌株中高度表达。虽然TgCDPK3调节寄生虫从宿主细胞的逸出、钙基浸润和组织囊肿的发育，但其调节的宿主靶蛋白仍不清楚。

方法

首先，使用质谱分析选择性结合TgCDPK3的蛋白质。随后，使用谷胱甘肽 - S - 转移酶（GST）下拉、免疫沉淀和免疫荧光测定来确认TgCDPK3与精氨酸酶 - 1之间的相互作用和共定位。进行蛋白质印迹和精氨酸酶活性测定以检测小鼠小胶质细胞系中内源性精氨酸酶 - 1和诱导型一氧化氮合酶（iNOS）的相对水平。进行荧光激活细胞分选（FACS）测定和酶联免疫吸附测定（ELISA）分析以确认在M1 / M2极化巨噬细胞内TgCDPK3与精氨酸酶 - 1之间相互作用的关联。进行细胞内增殖测定和噬斑测定以测试TgCDPK3与精氨酸酶 - 1之间的相互作用是否影响细胞间寄生虫生长。