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

Src 抑制可诱导弓形虫的选择性自噬杀伤，与 EGF 受体无关

• 批准号: 10679378
• 负责人: Alyssa Hubal
• 金额: $ 4.13万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-05-01 至 2026-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10679378
• 关键词: AKT inhibition; Address; Autophagocytosis; Autophagolysosome; Autophagosome; Binding Proteins; Biogenesis; Blindness; Cells; Chronic; Cyclic AMP-Dependent Protein Kinases; Data; Deposition; Disease; EGFR inhibition; Encephalitis; Endoplasmic Reticulum; Epidermal Growth Factor Receptor; Event; Eye; Eye diseases; Galactose Binding Lectin; Gefitinib; Impairment; In Vitro; Infectious Agent; Invaded; Lysosomes; Mediating; Membrane; Molecular; Mus; Ocular Toxoplasmosis; Outcome; PTK2 gene; Parasites; Parasitic infection; Patients; Population; Protein Kinase; Proteins; Resistance; Retina; Retinitis; Role; STAT3 gene; Signal Transduction; Signaling Molecule; Source; Testing; Therapeutic; Tissues; Toxoplasma gondii; Toxoplasmosis; Treatment outcome; Tyrosine Kinase Inhibitor; Ubiquitin; United States; Vacuole; Vision; Visual; Work; cell type; experimental study; improved; in vivo; inhibitor; knock-down; neglect; neural; new therapeutic target; novel; obligate intracellular parasite; parasite invasion; pathogen; prevent; receptor; recruit; recurrent infection; seropositive

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.

项目摘要 弓形虫（Toxoplasma gondii）是一种专性细胞内寄生虫，是眼弓形虫病的病原体 （OT）。这种慢性、复发性感染是全球感染性视网膜炎的首要原因， 25%的患者单眼失明。目前的治疗对视力结果没有积极影响。T. 弓形虫存在于寄生虫的空泡中，并诱导阻断自噬体-溶酶体的机制 在受感染的细胞中靶向寄生虫。寄生虫入侵后，T.弓形虫激活宿主细胞 信号分子Src，它驱动EGFR的延长的自磷酸化并激活EGFR的表达。 下游自噬抑制剂Akt。激活的Akt持续避免自噬靶向和存活， 牢房以前，EGFR抑制显示诱导T细胞的自噬杀伤。大约一半的 细胞，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 和EGFR的限制性表达。因此，Src可能是一个更好的靶点，因为它似乎可以激活Akt 其独立于EGFR，并且在神经组织和视网膜中广泛且高度表达。项目的目标是 理解T.在缺乏EGFR的细胞中， 自噬介导的寄生虫靶向，以了解Src抑制引发的分子事件 负责选择性靶向T.并确定Src是否抑制 防止OT。我们的初步研究表明Src基因的敲除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，Src抑制诱导自噬， 靶向似乎依赖于蛋白激酶的激活。因此，这个问题的核心假设 提出即使在没有EGFR的情况下，Src抑制也会杀死T.蛋白激酶依赖性弓形虫 自噬体的选择性靶向，Src抑制控制OT。目标1中提出的实验将 探讨Src在阻止T.在没有EGFR的情况下，在体外培养弓形虫。 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 Src抑制后的自噬体。目的3将探讨Src抑制对预先建立的OT的影响， vivo.这些数据将共同定义T.弓形虫抑制自噬靶向，解释如何 自噬选择性地靶向寄生虫，并可用于改善OT的治疗。