Autophagy and Ocular Toxoplasmosis

自噬和眼弓形虫病

• 批准号: 10391468
• 负责人: CARLOS S SUBAUSTE
• 金额: $ 37.65万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10391468
• 关键词: Animal Model; Antibodies; Autophagocytosis; Autophagosome; Blindness; Cells; Child; Confocal Microscopy; Deposition; Disease; EGF-Like Domain; EGFR inhibition; Elderly; Electron Microscopy; Epidermal Growth Factor Receptor; Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor; Event; Galactose Binding Lectin; Genetic; Impairment; Infection; Lead; Mediating; Membrane; Microbe; Molecular; Mus; Ocular Toxoplasmosis; Outcome; Parasites; Pathogenesis; Pathway interactions; Patients; Penetration; Pharmacology; Phosphorylation; Phosphotransferases; Process; Protein Kinase; Proteins; Recurrent disease; Relapse; Resistance; Retina; Retinitis; Role; STAT3 gene; Signal Pathway; Signal Transduction; Testing; Therapeutic; Toxoplasma gondii; Toxoplasmosis; Transactivation; Transgenic Mice; Ubiquitin; Vacuole; Vision; Visual; Work; congenital infection; immunosuppressed; improved; in vivo; inhibitor; live cell microscopy; novel; novel therapeutic intervention; pathogen; prevent; protective effect; protein activation; protein expression; recruit; therapeutic target

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.

细胞内原生动物弓形虫是传染性视网膜炎的最常见原因 世界。眼弓形虫病容易复发并导致 25% 的患者视力丧失，尤其是 先天性感染的儿童、老年人和免疫抑制者。目前的治疗确实 不能改善视功能或防止复发。更好地理解其机制 控制眼弓形体病可能会产生针对这种疾病的新治疗方法。 自噬是溶酶体降解的组成过程。弓形虫必须避免以 自噬以在宿主细胞内生存。我们发现，在入侵宿主细胞期间，弓形虫 诱导 EGFR 信号传导，从而避免最初的自噬靶向。最近，我们发现 弓形虫引起持续的 Src 信号传导，维持 EGFR 和 Akt（EGFR 的抑制剂）的激活 自噬）。 EGFR 的药物抑制触发先前弓形虫的自噬杀伤 感染细胞并预防眼弓形虫病。然而，保护是部分的（EGFR 表达受到限制； EGFR 仅部分负责 Akt 激活）。相比之下，Src 是 普遍存在的低浓度 Src 抑制剂可消除 Akt 激活并杀死弓形虫。如何 自噬体选择性靶向弓形虫（有效消除病原体所需）尚不清楚。 本申请的目的是检查 Src 在避免自噬杀伤中的作用 T. gondii，了解自噬如何选择性地针对寄生虫并确定其相关性 这种机制可以抵抗眼弓形虫病。中心假设是抑制 Src 能够激活特定的蛋白激酶，从而触发选择性自噬靶向和 杀死弓形虫，促进预防眼弓形虫病。在第一个目标中，我们将检查 Src 的抑制如何触发弓形虫感染细胞中该蛋白激酶的激活。这一目标将 使用阻断特定信号通路的遗传和药理学方法来追求。在 第二个目标是，我们将研究该激酶在弓形虫选择性自噬与大量自噬中的作用- 被感染的细胞。在第三个目标中，我们将检查由该蛋白激酶控制的分子事件 这解释了自噬体如何选择性地靶向寄生虫。这两个目标都将通过 使用针对内源性蛋白质、活细胞的抗体的共聚焦显微镜联合方法 使用荧光标记蛋白质和电子显微镜进行显微镜检查。在第四个目标中，我们将使用 眼弓形虫病和转基因小鼠的动物模型，用于检查 Src（蛋白质）的作用 眼弓形虫病中由 Src 和自噬控制的激酶。拟议的工作将进一步推动我们 了解宿主细胞信号传导如何调节弓形虫的自噬靶向及其结果 感染，并可能导致改善弓形体病治疗的辅助方法。