Blockade of host apoptosis by Toxoplasma gondii

弓形虫感染的细胞表现出深刻的凋亡抑制，表现在多个点 细胞凋亡级联。我们的研究表明软管转录因子NFKB在 寄生虫诱导的抗细胞凋亡状态的建立。多种细胞途径激活NFxB 通过其抑制剂kB上特定的Ser残基的磷酸化而发生。这种磷酸化事件是 由一个独特的细胞激酶复合体催化，该复合体定义了kB激酶信号体(Ikk)。在T.Gondii 感染细胞磷酸化kB定位于寄生液泡膜(PVM)，即细胞器 定义了允许细胞内复制的利基环境。无ALL细胞中P-kBat-PVM的检测 IKK活性表明寄生虫编码的激酶(TglKK)的存在是导致这一现象的原因。我们发现就是这样 寄生虫提取物和PVM富集组分中的一种活性。我们这里的重点是识别基因(S) 编码TglKK活性并检测其在NFxB激活和阻断细胞凋亡中的作用。 我们的数据表明，仅有TglKK活性不足以驱动NFKB基因在慢性粒细胞白血病细胞中的表达 IKK中的缺陷。对NFxB激活的时间性质的初步研究揭示了NFxB激活的双相模式 NFkB的表达提示宿主和寄生虫ikk的贡献是独立的，但在时间上是联系的 活动。我们建议在ikk上游的通路中使用具有特定损伤的宿主细胞系，以及 为了更好地描述相关的细胞通路，细胞系被“锁定”到一个已定义的表达谱中 被弓形虫感染颠覆。最后，我们开发了一种基因筛查来识别寄生虫基因 参与NFKB激活。初步结果证实，颠覆NFKB的机制是 多因素。这些寄生虫基因的鉴定及其在NFKB激活中的作用 对蜂窝组件的研究背景将有助于定义被 寄生虫。鉴于NFKB的许多目标，对这些通路的解剖尤为重要 细胞因子在急性弓形虫病发病机制和免疫发展中的作用 寄生虫。

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.