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.

细胞内的弓形虫是感染性视网膜炎的最常见原因 整个世界。眼弓形体病有复发的趋势，25%的患者会导致视力丧失，尤其是 在患有先天性感染的儿童中，老年人和免疫抑制者。目前的治疗方法是 不能改善视觉功能或防止复发。更好地理解 控制眼弓形体病可能会导致针对这种疾病的新的治疗方法。 自噬是溶酶体降解的一个结构性过程。弓形虫必须避免通过 自噬是为了在宿主细胞内生存。我们发现，在宿主细胞入侵期间，弓形虫 诱导EGFR信号，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，维持EGFR和Akt(抑制因子)的激活 自噬)。EGFR的药理抑制触发自噬杀灭弓形虫 感染细胞，预防眼弓形体感染。然而，保护是部分的(EGFR 表达受到限制；EGFR仅部分负责Akt的激活)。相比之下，Src是 普遍存在的低浓度的Src抑制剂可以抑制Akt的激活并杀死弓形虫。多么 自噬小体选择性地靶向弓形虫(有效消除病原体所需)尚不清楚。 本应用程序的目的是研究src在避免自噬杀伤中的作用 了解自噬是如何选择性地以寄生虫为目标的，并确定 这一机制在抵抗眼弓形虫病中起着重要作用。中心假说是，抑制 SRC能够激活特定的蛋白激酶，从而触发选择性的自噬靶向和 杀灭弓形虫促进对眼弓形虫病的保护。在第一个目标中，我们将检查 抑制Src如何在弓形虫感染细胞中触发该蛋白激酶的激活。这一目标将 通过阻断特定信号通路的遗传和药理学方法进行研究。在……里面 第二个目标，我们将研究该激酶在弓形虫选择性自噬与批量自噬中的作用。 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。 这解释了自噬是如何选择性地以寄生虫为目标的。这两个目标都将通过 利用抗内源蛋白、活细胞抗体的联合共聚焦显微镜方法 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 眼弓形体病动物模型和转基因小鼠检测蛋白Src的作用 眼弓形体病中Src调控的激酶与自噬拟议的工作将进一步推动我们的 了解宿主细胞信号如何调节弓形虫的自噬靶向及其结果 并可能导致采用辅助方法来改进弓形虫病的治疗。