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

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 弓形虫原虫是一种重要的人类和兽医病原体。这项建议的目的是了解弓形虫是如何干扰宿主细胞信号传递的，并确定对寄生虫生存的功能后果。病原体入侵宿主通常会激活核因子-kB家族 转录因子在免疫系统的调节中发挥重要作用，经常是抵抗感染所必需的。最近的研究表明，弓形虫入侵细胞不仅不能激活核因子-kB，而且这种寄生虫主动抑制这一信号通路，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，目前尚不清楚弓形虫是如何抑制核因子-kB的，以及重要的细胞靶点是什么。我们认为，当弓形虫感染巨噬细胞时，特定的弓形虫因子的存在与细胞质中的p65nf-kB蛋白有关。 掩盖它的核定位信号，防止它移位到核中。因此，在感染弓形虫的巨噬细胞中，依赖于核因子-kB的细胞因子的表达受到损害，因此，体内的适应性免疫反应也受到损害。在特定目标_1中，我们将确定 并确定了导致抑制核因子-kB信号通路的寄生因素。我们将专注于含有锚蛋白的TgEST 1207539蛋白，以及可能与p65 NF-kB相互作用的其他寄生虫蛋白，以及产生无法抑制NF-kB激活的寄生虫突变体。《特定目标2》 我们将研究弓形虫抑制核因子-kB的失败是否会恢复巨噬细胞在体外产生细胞因子的能力。我们将研究过度表达TgEST 1207539蛋白的巨噬细胞或感染寄生虫突变体的巨噬细胞产生的细胞因子。在特定的目标3中，我们将评估弓形虫突变体是否未能 抑制核因子-kB在体内感染时的毒力较小。这些研究结果将有助于确定核因子-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.