Src Inhibition Induces Selective Autophagic Killing of T. gondii Independently of EGF Receptor

项目总结 弓形虫是一种独有的细胞内寄生虫，也是眼弓形体病的致病原。 (OT)。这种慢性、反复感染是全世界感染性视网膜炎的首要原因，并可导致 25%的患者有一只眼失明。目前的治疗并没有对视力结果产生积极的影响。T. 弓形虫寄生在寄生液泡中，并诱导阻止自噬小体-溶酶体的机制。 在被感染的细胞中以寄生虫为靶标形成的。在寄生虫入侵后，弓形虫激活宿主细胞 信号分子Src，它驱动EGFR的长时间自动磷酸化，并激活 下游自噬抑制因子Akt.激活的Akt持续地避免自噬目标和存活 牢房。此前，抑制EGFR被证明可以诱导大约一半的弓形虫自噬死亡。 并对OT有部分保护作用。这种部分保护可能是部分Akt抑制的结果 EGFR的限制性表达。因此，Src可能是一个更好的目标，因为它似乎激活了Akt 不依赖于EGFR，在神经组织和视网膜中广泛和高表达。这个项目寻求 了解弓形虫在缺乏EGFR的细胞中利用的宿主信号机制，以防止 自噬介导的寄生虫靶向，以了解由Src抑制引发的分子事件 负责通过自噬选择性靶向弓形虫的基因，并确定是否抑制Src 保护您免受加班。我们的初步研究表明，敲除Src基因可以诱导细胞内寄生虫的杀伤 通过自噬介导的机制缺乏EGFR。此外，抑制Src可诱导自噬 靶向似乎依赖于蛋白激酶的激活。因此，这一点的中心假设 有人提出，即使在没有EGFR的情况下，Src的抑制也会由于依赖蛋白激酶而杀死弓形虫 自噬小体的选择性靶向和Src抑制控制OT。目标1中提出的实验将 研究在体外缺乏EGFR的情况下，Src在防止自噬杀灭弓形虫中的作用。 目标2中提出的实验将探索蛋白激酶激活在选择性寄生虫靶向中的作用。 通过Src抑制后的自噬小体。目的3探讨src抑制对脑内预先建立的OT的影响。 活着。这些数据将共同定义弓形虫抑制自噬靶向的机制，解释如何 自噬选择性地以寄生虫为靶标，可用于改进OT的治疗。

PROJECT SUMMARY Toxoplasma gondii is an obligate intracellular parasite and causative infectious agent of ocular toxoplasmosis (OT). This chronic, recurrent infection is the top cause of infectious retinitis worldwide and can lead to blindness in one eye in 25% of patients. Current treatment does not positively influence visual outcomes. T. gondii resides in a parasitophorous vacuole and induces mechanisms that block autophagosome-lysosome formation from targeting the parasite in infected cells. After parasite invasion, T. gondii activates the host cell signaling molecule, Src, which drives prolonged autophosphorylation of the EGFR and activates the downstream autophagy inhibitor, Akt. Activated Akt persistently avoids autophagic targeting and survival within the cell. Previously, EGFR inhibition was shown to induce autophagic killing of T. gondii in approximately half of the cells and is partially protective against OT. This partial protection may be a result of partial Akt inhibition and the restricted expression of EGFR. Thus, Src is likely a better target because it appears to activate Akt independently of EGFR and is broadly and highly expressed in neural tissue and the retina. This project seeks to understand the host signaling mechanism utilized by T. gondii in cells lacking EGFR that prevent autophagy-mediated targeting of the parasite, to understand the molecular events triggered by Src inhibition that are responsible for selective targeting of T. gondii by autophagy, and to determine whether Src inhibition protects against OT. Our preliminary studies demonstrate that knockdown of Src induces parasite killing in cells lacking EGFR through an autophagy-mediated mechanism. Additionally, Src inhibition induces autophagic targeting that appears dependent on the activation of a protein kinase. Therefore, the central hypothesis of this proposal is that, even in the absence of EGFR, Src inhibition kills T. gondii due to protein kinase-dependent selective targeting by autophagosomes, and Src inhibition controls OT. Experiments proposed in Aim 1 will investigate the role of Src in preventing the autophagic killing of T. gondii in the absence of EGFR in vitro. Experiments proposed in Aim 2 will explore the role of protein kinase activation in selective parasite targeting by autophagosomes after Src inhibition. Aim 3 will explore the effects of Src inhibition on pre-established OT in vivo. Together these data will define mechanisms in which T. gondii inhibits autophagic targeting, explain how autophagy selectively targets the parasite and may be applied to improved treatment for OT.