VERMONT COBRE: PROJECT 4: SUBVERSION OF HOST CELL SIGNALING BY TOXOPLASMA GOUDII

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。弓形虫是一种重要的人畜病原体。本提案的目的是了解弓形虫如何干扰宿主细胞信号并确定寄生虫存活的功能后果。病原体入侵宿主通常会激活NF-kB转录因子家族，这些转录因子在免疫系统的调节中起重要作用，并且通常需要抵抗感染。最近的研究表明，弓形虫入侵细胞不仅不能激活NF-kB，而且这种寄生虫积极抑制这一信号通路，使寄生虫能够在不触发促炎细胞因子诱导的情况下入侵细胞。然而，尚不清楚弓形虫是如何抑制NF-kB的，以及什么是重要的细胞靶点。我们认为，当弓形虫感染巨噬细胞时，一种特定的弓形虫因子的存在与细胞质中的p65 NF-kB蛋白相关，该蛋白掩盖了其核定位信号并阻止其易位到核中。因此，受感染巨噬细胞中响应弓形虫的nf - kb依赖性细胞因子的表达受损，因此，体内的适应性免疫反应也受损。在Specific aim_1中，我们将鉴定和表征负责抑制NF-kB信号通路的寄生虫因子。我们将重点关注含有锚蛋白的TgEST 1207539蛋白，其他可能与p65 NF-kB相互作用的寄生虫蛋白，以及产生不能抑制NF-kB激活的寄生虫突变体。在特异性目的2中，我们将研究弓形虫抑制NF-kB的失败是否能恢复巨噬细胞在体外产生细胞因子的能力。我们将研究过表达TgEST 1207539蛋白或感染寄生虫突变体的巨噬细胞的细胞因子产生。在特异性目标3中，我们将评估未能抑制NF-kB的弓形虫突变体在体内感染中的毒性是否较低。这些研究的结果将有助于确定NF-kB信号通路在弓形虫免疫应答中的功能意义。这项研究将对这种寄生虫用来操纵宿主细胞信号的分子机制产生新的见解。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The protozoan parasite Toxoplasma gondii is an important human and veterinary pathogen. The objective of this proposal is to understand how T. gondii interferes with host cell signaling and determine the functional consequences for parasite survival. Invasion of a host by a pathogen usually activates the NF-kB family of transcription factors which play an important role in the regulation of the immune system and are frequently required for resistance to infection. Recent studies have shown that invasion of cells by T. gondii not only fails to activate NF-kB, but this parasite actively inhibits this signaling pathway, enabling the parasite to invade cells without triggering proinflammatory cytokine induction. However, it remains unclear how T. gondii inhibits NF-kB and what is the important cellular target. We propose that when T. gondii infect macrophages the presence of a specific T. gondii factor associates with a p65 NF-kB protein in the cytoplasm masking its nuclear localization signals and preventing its translocation into the nucleus. As a result, expression of NF-kB-dependent cytokines in response to T. gondii in infected macrophages is impaired and, as a consequence, the adaptive immune response is also impaired in vivo. In Specific Aim_ 1 we will identify and characterize parasite factors that are responsible for the inhibition of the NF-kB signaling pathway. We will focus on an ankyrin-containing TgEST 1207539 protein, other parasite proteins that may interact with p65 NF-kB, and on generating parasite mutants that fail to inhibit the activation of NF-kB. In Specific Aim 2 we will examine if failure of T. gondii to inhibit NF-kB restores the ability of macrophages to produce cytokines in vitro. We will study cytokine production of macrophages overexpressing the TgEST 1207539 protein or infected with parasite mutants. In Specific Aim 3 we will evaluate if T. gondii mutants that fail to inhibit NF-kB are less virulent in an in vivo infection. The outcome of these studies will be useful to determine the functional significance of the NF-kB signaling pathway for the immune response to T. gondii. This research will yield new insights into the molecular mechanisms that this parasite uses to manipulate the host cell signaling.