Autophagy and Ocular Toxoplasmosis

细胞内的弓形虫是感染性视网膜炎最常见的原因， 世界眼弓形虫病往往会复发，并导致25%的患者视力丧失，特别是 儿童先天性感染、老年人及免疫抑制者。目前的治疗方法 不能改善视觉功能或防止复发。更好地理解 控制眼弓形虫病可能会导致新的治疗方法对这种疾病。 自噬是溶酶体降解的组成性过程。T.弓形虫必须避免靶向， 自噬以在宿主细胞内存活。结果表明，T.弓形虫 诱导EGFR信号传导，导致避免初始自噬靶向。最近，我们发现 这个T。弓形虫引起持续的Src信号传导，维持EGFR和Akt的活化（ 自噬）。EGFR的药理学抑制触发T.弓形虫在以前 感染的细胞，并防止眼部弓形虫病。然而，保护是部分的（EGFR 表达受到限制; EGFR仅部分负责Akt活化）。相比之下，Src 普遍存在的低浓度Src抑制剂消除Akt活化并杀死T.刚地。如何 自噬体选择性靶向T.弓形虫（有效消除病原体所需）不详。 本申请的目的是检查Src在避免自噬性杀伤中的作用， T.了解自噬如何选择性地靶向寄生虫，并确定 这一机制对眼弓形虫病的抵抗力。核心假设是， Src能够激活特定的蛋白激酶，触发选择性自噬靶向， 杀死T。弓形虫促进对眼部弓形虫病的保护。在第一个目标中，我们将研究 Src的抑制如何触发T.感染了生殖腺的细胞这一目标将 使用阻断特定信号通路的遗传和药理学方法进行。在 第二个目的是研究这种激酶在T.弓形虫， 被感染的细胞在第三个目标中，我们将研究由这种蛋白激酶控制的分子事件 解释了自噬体如何选择性地攻击寄生虫这两个目标将通过一个 使用抗内源性蛋白质抗体的共聚焦显微镜的组合方法，活细胞 使用荧光标记蛋白质的显微镜和电子显微镜。在第四个目标中，我们将使用 眼弓形虫病的动物模型和转基因小鼠，以检查Src蛋白的作用， 眼弓形虫病中Src控制的激酶和自噬拟议的工作将进一步促进我们的 理解宿主细胞信号传导如何调节T.弓形虫及其结局 并可能导致预防性的方法来改善弓形虫病的治疗。

The intracellular protozoan Toxoplasma gondii is the most common cause of infectious retinitis in the world. Ocular toxoplasmosis tends to recur and leads to vision loss in 25% of patients, especially in children with congenital infection, the elderly and the immunosuppressed. Current treatment does not improve visual function or prevent relapses. A better understanding of the mechanisms that control ocular toxoplasmosis may result in novel therapeutic approaches against this disease. Autophagy is a constitutive process of lysosomal degradation. T. gondii must avoid targeting by autophagy in order to survive within host cells. We showed that during invasion of host cells, T. gondii induces EGFR signaling that results in avoidance of initial autophagic targeting. Recently, we found that T. gondii causes sustained Src signaling that maintains activation of EGFR and Akt (inhibitor of autophagy). Pharmacologic inhibition of EGFR triggers autophagic killing of T. gondii in previously infected cells and protects against ocular toxoplasmosis. However, the protection is partial (EGFR expression is restricted; EGFR is only partially responsible for Akt activation). In contrast, Src is ubiquitous and low concentrations of a Src inhibitor ablates Akt activation and kills T. gondii. How autophagosomes selectively target T. gondii (required for effective pathogen elimination) is unknown. The objective of this application is to examine the role of Src in avoidance of autophagic killing of T. gondii, understand how autophagy selectively targets the parasite and determine the relevance of this mechanism in resistance against ocular toxoplasmosis. The central hypothesis is that inhibition of Src enables the activation of a specific protein kinase that triggers selective autophagic targeting and killing of T. gondii promoting protection against ocular toxoplasmosis. In the first aim we will examine how inhibition of Src triggers activation of this protein kinase in T. gondii-infected cells. This aim will be pursued using genetic and pharmacologic approaches that block specific signaling pathways. In the second aim we will examine the role of this kinase in selective vs bulk autophagy in T. gondii- infected cells. In the third aim, we will examine the molecular events controlled by this protein kinase that explain how autophagosomes selectively target the parasite. Both aims will be pursued using a combined approach of confocal microscopy using antibodies against endogenous proteins, live-cell microscopy using fluorescently-tagged proteins and electron microscopy. In the fourth aim we will use an animal model of ocular toxoplasmosis and transgenic mice to examine the role of Src, the protein kinase controlled by Src and autophagy in ocular toxoplasmosis. The proposed work will further our understanding of how host cell signaling regulates autophagic targeting of T. gondii and the outcome of the infection, and may lead to adjunctive approaches to improve the treatment of toxoplasmosis.