Identification of Toxoplasma genes that mediate its colonization of the eye

鉴定介导其在眼睛定植的弓形虫基因

• 批准号: 10040382
• 负责人: JEROEN SAEIJ
• 金额: $ 23.55万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-01 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10040382
• 关键词: Adrenal Cortex Hormones; Affect; Anatomy; Antibiotics; Blindness; Blood; Blood-Retinal Barrier; CRISPR screen; CRISPR/Cas technology; Candidate Disease Gene; Cells; Chorioretinitis; Chronic; Cicatrix; Complement; Congenital Toxoplasmosis; Consensus; Cyst; Cytolysis; Cytoplasm; Data; Defect; Dendritic Cells; Development; Disease; Distant; Epithelial Cells; Equus caballus; Estrogen receptor positive; Etiology; Eye; Eye Development; Eye diseases; Genes; Goals; Human; Imaging Techniques; Immune; Immunocompetent; In Vitro; Individual; Infection; Inflammation; Knock-out; Lead; Lesion; Leukocytes; Literature; Locomotion; Luciferases; Mediating; Mus; Ocular Toxoplasmosis; Organ; Outcome; Parasites; Patients; Pharmaceutical Preparations; Phenotype; Posterior Uveitis; Prevention; Process; Proteins; Publishing; Pyrimethamine; Recurrence; Regimen; Research; Resolution; Retina; Shuttle Vectors; Site; Testing; Therapeutic; Time; Tissues; Toxoplasma; Toxoplasma gondii; Uveitis; acute infection; base; design; follow-up; high reward; high risk; in vivo; in vivo imaging; inhibitor/antagonist; knockout gene; loss of function; migration; novel; novel strategies; novel therapeutics; obligate intracellular parasite; ocular imaging; prevent; side effect; whole body imaging

The obligate intracellular parasite Toxoplasma gondii causes ocular disease in 70-90% of congenital toxo- plasmosis patients. Additionally, ocular toxoplasmosis (OT) characterized by toxoplasmic retinochoroiditis can also follow acute infection and is the most common cause of infectious uveitis in immunocompetent and im- munocompromised individuals. Disease is generally self-limiting; however, recurrent lesions are frequent. Res- olution of active lesions leads to tissue scarring, with loss of anatomical and functional integrity of the retina that can lead to permanent visual loss. There is no consensus regarding treatment of OT. Therapeutic regi- mens usually include antibiotics like pyrimethamine and sulfamethazone, combined or not with systemic corti- costeroids. Our long-term goal is to help identify novel T.gondii proteins that could be targeted with novel drugs. These are urgently needed since current drugs have toxic side effects, especially during prolonged treatment which is necessary during recurrent OT, and they do not clear the chronic cyst stages. To cause OT it is likely that T.gondii needs to disseminate to the eye and breach the blood-retinal barri- er. Parasite-mediated cell lysis can cause tissue damage; however, exacerbated inflammation is also an im- portant cause of tissue destruction. The overall objectives in this application are to determine how T.gondii reaches the eye, breaches the blood-retinal barrier and subsequently promotes development of ocular inflam- mation. T.gondii secretes effector proteins into the host cytoplasm to co-opt host cells. T.gondii-infected den- dritic cells (DCs) display hypermotility and infected DCs have enhanced transmigration across retinal epithelial cells. The central hypothesis is that T.gondii co-opts immune cells, such as DCs, as Trojan Horses to dissemi- nate to the eye and to transmigrate across the blood-retinal-barrier. Using a T.gondii CRISPR/Cas9 loss-of- function screen in mice, our lab has identified multiple T.gondii candidate genes that appear to affect its ability to colonize the eye without affecting replication efficiency. In the first aim it will be confirmed if knocking out T.gondii candidate genes that appear involved in colonization of the eye based on the CRISPR screen have a defect in colonization of the eye of infected mice. In vivo parasite replication, dissemination, colonization of the eye, and ocular inflammation, will be compared between knockout and wild-type parasites using multiple in vi- vo whole body and ocular imaging techniques on live mice over time. In the second aim an in vitro CRISPR/ Cas9 loss-of-function screen will be used to identify T.gondii genes that mediate infected leukocyte migration across polarized human retinal epithelial cells. For parasite knockouts strains generated in Aim 1, it will also be tested if they have a defect in using DCs to cross polarized retinal epithelial cells. The proposed research is significant because identification of novel T.gondii genes that determine colonization of the eye and ocular dis- ease has the potential to help with the rational design of novel therapies to treat ocular toxoplasmosis.

专性细胞内寄生虫弓形虫会导致 70-90% 先天性弓形虫患者眼部疾病 浆虫病患者。此外，以弓形虫性视网膜脉络膜炎为特征的眼弓形虫病（OT）可以 也发生在急性感染之后，并且是免疫功能健全和免疫力低下的感染性葡萄膜炎的最常见原因。 免疫功能低下的个体。疾病一般具有自限性；然而，复发性病变很常见。雷斯- 活动性病变的消退导致组织疤痕，视网膜解剖结构和功能完整性丧失 这可能会导致永久性视力丧失。关于 OT 的治疗尚未达成共识。治疗方案 男性通常使用乙胺嘧啶和磺胺二甲酮等抗生素，与全身性皮质激素联合或不联合使用。 类固醇。我们的长期目标是帮助识别新的弓形虫蛋白，这些蛋白可以作为新的目标 药物。这些都是迫切需要的，因为目前的药物具有毒副作用，特别是在长期治疗期间 治疗在复发性 OT 期间是必要的，并且不能清除慢性囊肿阶段。 为了引起 OT，弓形虫可能需要传播到眼睛并突破血-视网膜屏障。 呃。寄生虫介导的细胞裂解会导致组织损伤；然而，炎症加剧也是一个重要因素 组织破坏的重要原因。本应用的总体目标是确定弓形虫如何 到达眼睛，突破血视网膜屏障，随后促进眼部炎症的发展 化。弓形虫将效应蛋白分泌到宿主细胞质中以选择宿主细胞。弓形虫感染的 Den- 树突状细胞 (DC) 表现出过度运动，并且受感染的 DC 增强了跨视网膜上皮的迁移 细胞。中心假设是弓形虫利用免疫细胞（例如树突状细胞）作为特洛伊木马来传播 天然存在于眼睛中并穿过血-视网膜屏障。使用弓形虫 CRISPR/Cas9 缺失- 在小鼠的功能筛选中，我们的实验室已经鉴定出多个弓形虫候选基因，这些基因似乎会影响其能力 在不影响复制效率的情况下定植眼睛。在第一个目标中，如果击倒将被确认 根据 CRISPR 筛选，弓形虫候选基因似乎参与了眼睛的定植，其中有一个 受感染小鼠眼睛定植缺陷。寄生虫在体内的复制、传播、定植 眼睛和眼部炎症，将使用多重体内比较，敲除型寄生虫和野生型寄生虫 vo 随着时间的推移，活体小鼠的全身和眼部成像技术。第二个目标是体外 CRISPR/ Cas9 功能丧失筛选将用于鉴定介导受感染白细胞迁移的弓形虫基因 穿过极化的人视网膜上皮细胞。对于目标 1 中生成的寄生虫敲除菌株，也将 测试他们在使用树突状细胞穿过极化的视网膜上皮细胞时是否存在缺陷。拟议的研究是 意义重大，因为确定了决定眼睛定植和眼部疾病的新弓形虫基因 easy 有可能帮助合理设计治疗眼弓形虫病的新疗法。