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Blockade of host apoptosis by Toxoplasma gondii

弓形虫阻断宿主细胞凋亡

基本信息

批准号：
7766941
负责人：
ANTHONY P. SINAI
金额：
$ 31.08万
依托单位：
UNIVERSITY OF KENTUCKY
依托单位国家：
美国
项目类别：
财政年份：
2001
资助国家：
美国
起止时间：
2001-03-01 至 2012-02-28
项目状态：
已结题

项目摘要

Toxoplasma gondii infected cells exhibit a profound blockade of apoptosis that manifests at multiple points in the apoptotic cascade. Our studies have shown an essential role for the hose transcription factor NFKB in the establishment of the parasite-directed anti-apoptotic state. Activation of NFxB by diverse cellular pathways occurs via the phosphorylation of specific Ser residues on its inhibitor kB. This phosphorylation event is catalyzed by a unique cellular kinase complex defining the kB kinase signalosome (IKK). In T.gondii infected cells Phospho-kB localizes at the parasitophorous vacuole membrane (PVM), the oraganelle defining the intracellular replication-permissive niche. The detection of P-kBat the PVM in cells devoid of all IKK activity suggested the presence of a parasite-encoded kinase (TglKK) is responsible. We find just such an activity in parasite extracts and PVM-enriched fractions. We focus here on identifying the gene(s) encoding the TglKK activity and examining its role both in NFxB activation and the blockade of apoptosis. Our data indicate that TglKK activity alone is not sufficient to drive NFKB gene expression in cells with defects in IKK. Initial studies examining the temporal nature of NFxB activation reveal a biphasic pattern of NFKB expression suggesting independent, but temporally linked, contributions of the host and parasite IKK activities. We propose to use host cell lines with specific lesions in patheways upstream of IKK, as well as cell lines "locked" into a defined expression profile, to better characterize the pertinent cellular pathways subverted by T.gondii infection. Finally, we have developed a genetic screen to identify parasite genes involved in NFKB activation. Initial results bear out the evidence that the mechanism to subvert NFKB is multifactorial. Identification of these parasite genes and the elucidation of their roles in NFKB activation in the context of the studies on the cellular components will help define the signaling networks subverted by the parasite. Dissection of these pathways is particularly important given the targets of NFKB many of the cytokines implicated both in the pathogenesis of acute toxoplasmosis and the development of immunity to the parasite.

弓形虫感染的细胞表现出对凋亡的深度阻断，这表现在多个点，凋亡级联反应我们的研究已经显示了软管转录因子NF κ B在细胞凋亡中的重要作用。建立寄生虫导向的抗凋亡状态。NFxB通过不同的细胞途径激活通过其抑制剂kB上特定Ser残基的磷酸化发生。这种磷酸化作用是由定义kB激酶信号体（IKK）的独特细胞激酶复合物催化。在弓形虫感染细胞磷酸化kB定位于寄生虫空泡膜（PVM），定义了细胞内允许复制的生态位。在PVM中检测P-kB在缺乏所有 IKK活性表明寄生虫编码的激酶（TglKK）的存在是负责的。我们发现在寄生虫提取物和富含PVM的级分中的活性。我们在这里集中于识别基因（S）编码TglKK活性并检测其在NF xB活化和细胞凋亡阻断中的作用。我们的数据表明，单独的TglKK活性不足以驱动NF κ B基因在细胞中的表达， IKK的缺陷。检查NF xB激活的时间性质的初步研究揭示了NF xB激活的双相模式。 NF κ B表达提示宿主和寄生虫IKK的独立但时间相关的贡献活动我们建议使用在IKK上游通路中具有特异性病变的宿主细胞系，以及将细胞系“锁定”到确定的表达谱中，以更好地表征相关的细胞途径被弓形虫感染破坏最后，我们开发了一种基因筛选技术，参与NF κ B活化。初步结果证实了破坏NFKB的机制是多因素的鉴定这些寄生虫基因并阐明它们在NFkB活化中的作用。对细胞成分的研究背景将有助于定义被破坏的信号网络。寄生虫考虑到NF κ B的靶点，这些途径的解剖是特别重要的。急性弓形虫病的发病机制和对弓形虫病的免疫发展都涉及细胞因子。寄生虫