弓形虫CDPK3调控宿主巨噬细胞偏移在弱毒株免疫逃逸中的机制研究

弓形虫II型弱毒株是感染人类的主要虫株，易导致慢性隐性感染并在人群中广泛传播，开展弱毒株与宿主相互作用的研究，可揭示其免疫逃逸新机制。在弓形虫感染的固有免疫应答阶段，巨噬细胞(Mφ)向M1极化可控制虫体过度繁殖，使宿主进入慢性隐性感染状态。前期我们发现TgCDPK3在II型和Wh6弱毒株高表达，可与宿主IKK相互作用，促进IκB磷酸化和降解，激活NF-κB信号通路，诱导Mφ向M1极化，提示TgCDPK3是一个重要的免疫逃逸虫体效应分子。本项目构建敲除Tgcdpk3的各种虫株和其重组慢病毒，体内外研究其对Mφ极化的影响；以TgCDPK3/IKK相互作用和磷酸化为切入点，着重阐明TgCDPK3调控NF-κB的关键分子及翻译后修饰的精确分子机制；探讨以TgCDPK3为靶点的突变体和小分子抑制剂/激活剂逆转Mφ极化，为防治弓形虫病提供实验依据,也为我国流行的Wh6弱毒株成囊及隐性感染提供新机制。

Toxoplasma gondii is a foodborne parasitic protozoon that can infect virtually all warm-blooded animals, including humans. Type II strains are most commonly associated with human infections in both cases of congenital infection and immunocompromised patients, and Type II strains are the most seen in humans. Different strains of Toxoplasma have evolved specific effector molecules to modulate the immune responses of different hosts. Type I and III strains infected macrophages, a cell type required for host immunity to Toxoplasma, are alternatively activated (M2), while type II infected macrophages are classically activated (M1). Understanding the basis of Toxoplasma strain differences and how effectors work to achieve immunologic escape is a major goal of current research. TgCDPK3, belongs to the superfamily of kinases, which restricted to the apicomplexans, ciliates, and plants. It has been documented that TgCDPK3 is localized to the parasite periphery in intracellular and extracellular parasites. Our preliminary data indicated that TgCDPK3 was highly expressed in less virulent II and Chinese 1 Wh6 strains. Strikingly, GST-pull down revealed that TgCDPK3 directly interacted with IKKα. TgCDPK3 over-expression facilitated the phosphorylation and degradation of IκBα. Futhermore, double luciferase reporter assay showed that TgCDPK3 over-expression increased NF-κB activity and induced macrophages to polarize toward M1. Our results strongly suggested that TgCDPK3 might be another important effector molecule inducing macrophage polarization, but the mechanism of regulating immune response is unclear. The present study will analyze the polarization of macrophages infected with ME49cdpk3-/- and WH6cdpk3-/- knockout transgenic strains. In addition, we will uncover the signal pathway and illuminate the mechanism. Therefore, our central hypothesis is that the interaction between TgCDPK3 and IKKα facilitates phosphorylation and degradation of IκBα to activate NF-κB signal transduction. We propose to define the interaction domain of TgCDPK3/IKKα and illuminate the mechanisms by using recombinant TgCDPK3 viruses and ME49cdpk3-/- and WH6 cdpk3-/- knockout transgenic strains. The systematic studies proposed in this project will reveal the novel Toxoplasma effectors that modulate macrophage activation and affect the ability of Toxoplasma to establish a chronic infection, and provide experiment basis of targeted therapy for toxoplasmosis.