Blockade of host apoptosis by Toxoplasma gondii

描述（由申请方提供）：弓形虫感染的细胞表现出对细胞凋亡的深度阻断，其在细胞凋亡级联反应中的多个点表现出来。我们的研究表明，软管转录因子NF κ B在建立寄生虫导向的抗凋亡状态的重要作用。不同细胞途径对NFxB的激活通过其抑制剂kB上的特异性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。于T.弓形虫感染的细胞磷酸-kB定位于寄生虫空泡膜（PVM），该细胞器限定了细胞内允许复制的小生境。在没有IKK活性的细胞中检测到PVM处的P-kB表明寄生虫编码的激酶（TglKK）的存在是负责的。我们在寄生虫提取物和富含PVM的馏分中发现了这样的活性。我们在此集中于鉴定编码TglKK活性的基因，并研究其在NFxB激活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IKK缺陷细胞中NF κ B基因的表达。检查NF xB激活的时间性质的初步研究揭示了NF κ B表达的双相模式，表明宿主和寄生虫IKK活性的独立但时间相关的贡献。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 细胞系“锁定”到一个确定的表达谱，以更好地表征相关的细胞通路颠覆T。弓形虫感染最后，我们开发了一种遗传筛选来鉴定参与NF κ B活化的寄生虫基因。初步结果证实破坏NF κ B的机制是多因素的。这些寄生虫基因的鉴定和阐明其在NF κ B活化中的作用的背景下，对细胞组分的研究将有助于确定寄生虫破坏的信号传导网络。考虑到NF κ B的靶点，这些途径的解剖是特别重要的，NF κ B是涉及急性弓形虫病的发病机制和对寄生虫的免疫力的发展的许多细胞因子。

DESCRIPTION (provided by applicant): 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 organelle defining the intracellular replication-permissive niche. The detection of P-kB at 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 pathways 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.