Signal transduction during Toxoplasma infection

描述：(申请人提供)：这笔赠款的长期目标 在了解弓形虫分子致病机制中的应用 感染。这项建议侧重于巨噬细胞内的信号通路。 在弓形虫感染期间。巨噬细胞是重要的宿主细胞。 感染，尽管它们是炎症性疾病的有效来源 当被微生物刺激激活时，如细菌 脂多糖(LPS)。最近，我们发现寄生虫感染 巨噬细胞抑制NFkappaB依赖的细胞因子 肿瘤坏死因子-α和白介素12。其实现机制是由于 寄生虫诱导的IkcappaB降解的阻断，后者是 启用NFkappaB核转位所必需的。这样做的目的是 建议对这一现象进行详细研究，并考察 感染其他信号通路和宿主细胞。有四个具体的 旨在得到解决。1、确定感染干扰的机制 通过内毒素触发的NFkappaB激活途径，并确定哪个 这种信号级联的组件由寄生虫触发。这将是 用免疫化学技术评估IkappaB 泛素化，以及利用显性负突变体进行的转染实验 位于IkappaB磷酸化上游的信号分子。2、确定 MAPK家族成员在速殖子诱导的肿瘤坏死因子-α阻断中的作用 IL-12，没有生产。这将通过结合以下几个方面来实现 磷酸特异性免疫印迹，并对每个主要MAPK进行活性分析。 3、确定寄生虫诱导的抑制是否通过基因减少而发生 转录或增加了mRNA的稳定性，使用核连续分析和RPA， 分别进行了分析。生物阵列技术也将用于识别其他 弓形虫感染上调或下调巨噬细胞基因。4、确定 当细胞内寄生虫被感染时，巨噬细胞是否保持内毒素无反应 被杀死，以及当细胞通过吞噬将寄生虫内化。另外，确定 弓形虫是否抑制树突状细胞和肠道功能 上皮细胞对内毒素的反应。这些目标将通过以下方式实现 共聚焦荧光显微镜和电泳迁移率改变分析 检测NFkappaB核转位。这项建议与健康有关 弓形虫是一种免疫受损的主要机会性感染 患者群体，以及严重的先天性感染。了解 感染的分子病理学将导致更好的控制策略 这种病原体和相关的微生物病原体。

DESCRIPTION: (provided by the applicant): The long-term objective of this grant application is to understand the molecular pathogenesis of Toxoplasma gondii infection. This proposal focuses on macrophage intracellular signaling pathways during T. gondii infection. Macrophages serve as important host cells during infection, despite the fact that they are a potent source of inflammatory mediators when activated by microbial stimuli such as bacterial lipopolysaccharide (LPS). Recently, we found that parasite infection of macrophages results in suppression of the NFkappaB-dependent cytokines TNF-alpha and IL- 12. The mechanism by which this is achieved is due to a parasite-induced blockade in IkcappaB degradation, the latter of which is required to enable NFkappaB nuclear translocation. The objective of this proposal is to examine this phenomenon in detail, and to examine the effect of infection on other signaling pathways and host cells. There are four specific aims to be addressed. 1, Determine the mechanism by which infection interferes with the LPS-triggered NFkappaB activation pathway, and determine which components of this signaling cascade are triggered by the parasite. This will be accomplished employing immunochemical techniques to evaluate IkappaB ubiquitination, and transfection experiments using dominant negative mutants of signaling molecules lying upstream of IkappaB phosphorylation. 2, Determine the involvement of MAPK family members in tachyzoite-induced blockade of TNF-alpha, IL- 12, and NO production. This will be achieved by a combination of phospho-specific immunoblotting, and activity assays on each of the major MAPK. 3, Determine whether parasite-induced inhibition occurs through decreased gene transcription or increased mRNA stability, using nuclear run-on assays and RPA, respectively. Bio-array technology will also be employed to identify other macrophage genes up- or down-regulated by Toxoplasma infection. 4, Determine whether macrophages remain LPS non-responsive when intracellular parasites are killed, and when cells internalize parasites by phagocytosis. Also, determine whether Toxoplasma inhibits the ability of dendritic cells and intestinal epithelial cells to respond to LPS. These objectives will be accomplished using confocal fluorescence microscopy and electrophoretic mobility shift assay to detect NFkappaB nuclear translocation. The health relatedness of this proposal is that Toxoplasma is a major opportunistic infection in immunocompromised patient populations, and a serious congenital infection. Understanding the molecular pathology of infection will lead to better control strategies for this and related microbial pathogens.