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: 10242167
• 负责人: JEROEN SAEIJ
• 金额: $ 19.04万
• 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 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10242167
• 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; 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.

强制性细胞内寄生虫弓形虫弓形虫在70-90％的先天性毒品中引起眼科疾病 血浆患者。另外，以弓形虫性视网膜毒性炎为特征的眼癌（OT）可以 还遵循急性感染，是免疫能力和不受欢迎的感染性葡萄膜炎的最常见原因 Munocompromprompromist的个人。疾病通常是自我限制的；但是，经常发生复发性病变。 res- 活性病变的溶解导致组织疤痕，视网膜的解剖和功能完整性丧失 这可能导致永久视觉损失。关于OT的治疗没有共识。治疗治疗 男士通常包括丙烯胺和磺胺甲酮等抗生素，与全身性皮质合并 类coldoid。我们的长期目标是帮助识别可用于新颖的新型T.gondii蛋白 毒品。由于当前药物具有有毒的副作用，尤其是在长时间的时间里，因此迫切需要这些 在复发期间必不可少的治疗，并且无法清除慢性囊肿阶段。 为了引起ot，t.gondii可能需要向眼睛传播并违反血液视网膜barri- 嗯。寄生虫介导的细胞裂解会导致组织损伤。但是，加剧的炎症也是一种 组织破坏的田径原因。本应用程序中的总体目标是确定t.gondii 到达眼睛，破坏血视网膜屏障，随后促进了眼睛的发展 mation。 T.gondii将效应蛋白分泌到宿主细胞质中，以相互宿主细胞。 t.gondii感染的den- Dritic细胞（DC）表现出高电动性，被感染的DC在视网膜上皮跨移民增强 细胞。中心假设是T.gondii合作的免疫细胞（例如DCS），例如特洛伊木马去散开 围绕着眼睛，跨血视视视网膜级迁移。使用t.gondii crispr/cas9丧失 在小鼠中的功能屏幕，我们的实验室已经确定了多个T.Gondii候选基因，这些基因似乎影响了其能力 在不影响复制效率的情况下定居眼睛。在第一个目标中，如果敲除 基于CRISPR屏幕的t.gondii候选基因的眼睛具有 感染小鼠眼睛的定植缺陷。体内寄生虫复制，传播，定殖 眼睛和眼部炎症将在敲除和野生型寄生虫之间进行比较 随着时间的推移，活体小鼠的全身和眼部成像技术。在第二个目标中，体外CRISPR/ CAS9功能丧失屏幕将用于识别介导感染性白细胞迁移的T.gondii基因 跨过极化的人类视网膜上皮细胞。对于在AIM 1中产生的寄生虫敲除菌株，也将是 测试是否在使用DC跨偏振视网膜上皮细胞方面有缺陷。拟议的研究是 显着，因为确定了确定眼睛和眼部定植的新型T.gondii基因 轻松的潜力有可能帮助治疗眼毒的新型疗法的合理设计。