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引起70-90%的先天性弓形虫眼病， 胞浆菌病患者此外，以弓形体视网膜脉络膜炎为特征的眼弓形体病（OT）可 也是急性感染后，是免疫活性和免疫缺陷患者感染性葡萄膜炎的最常见原因。 免疫功能低下的人疾病通常是自限性的;然而，复发性病变是常见的。Res- 活动性病变的消退导致组织瘢痕形成，视网膜的解剖和功能完整性丧失 会导致永久性视力丧失关于OT的治疗没有共识。治疗区域 男性通常包括乙胺嘧啶和磺胺甲恶唑等抗生素，无论是否与全身性皮质激素联合使用， 类固醇我们的长期目标是帮助鉴定新的弓形虫蛋白， 毒品这些都是迫切需要的，因为目前的药物有毒副作用，特别是在长期 治疗，这是必要的，在复发OT，他们不清楚慢性囊肿阶段。 要引起OT，弓形虫很可能需要传播到眼睛并突破血视网膜屏障， 儿寄生虫介导的细胞溶解可导致组织损伤;然而，炎症加剧也是一种免疫反应。 组织破坏的重要原因本申请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， mation。刚地弓形虫分泌效应蛋白进入宿主细胞质以同化宿主细胞。刚地弓形虫感染的巢穴 树突状细胞（DCs）表现出高运动性，并且感染的DCs具有增强的跨视网膜上皮的迁移 细胞中心假设是弓形虫选择免疫细胞，如DC，作为特洛伊木马来传播， 进入眼睛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，我们的实验室已经确定了多个弓形虫候选基因，这些基因似乎会影响其功能， 在不影响复制效率的情况下定殖于眼睛。在第一个目标中，如果击倒，将得到确认 基于CRISPR筛选，似乎参与眼睛定殖的弓形虫候选基因具有与眼相关的基因。 感染小鼠眼睛定植缺陷。体内寄生虫复制、传播、 眼睛和眼部炎症，将使用多个invi- VO全身和眼部成像技术随时间对活小鼠的影响。在第二个目标中，体外CRISPR/ Cas9功能丧失筛选将用于鉴定介导感染的白细胞迁移的弓形虫基因 穿过极化的人类视网膜上皮细胞。对于Aim 1中产生的寄生虫敲除菌株，还将 测试他们是否在使用DC交叉极化视网膜上皮细胞方面存在缺陷。拟议的研究是 重要的是，因为确定新的弓形虫基因，决定殖民的眼睛和眼disc， EASY具有帮助合理设计治疗眼部弓形虫病的新疗法的潜力。