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.

弓形虫专性细胞内寄生虫可导致70%-90%的先天性弓形虫患者的眼部疾病。 血浆病患者。此外，以弓形体视网膜脉络膜炎为特征的眼弓形体病(OT)可以 其次是急性感染，是感染性葡萄膜炎的最常见原因，在免疫功能和免疫不全方面。 免疫受损的人。疾病一般是自限性的；然而，复发的病变很常见。资源- 活动性病变的解决会导致组织瘢痕形成，并导致视网膜的解剖和功能完整性丧失。 这可能会导致永久性的视力丧失。对于OT的治疗，目前尚无共识。治疗规则- 男性通常包括乙胺嘧啶和磺胺二甲基嘧啶等抗生素，无论是否与全身皮质醇联合使用。 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 毒品。这些都是迫切需要的，因为目前的药物有毒副作用，特别是在长期服药期间 在复发的OT期间进行必要的治疗，并且不能清除慢性囊肿期。 为了引起OT，弓形虫很可能需要传播到眼睛并突破血-视网膜屏障-- 呃。寄生虫介导的细胞裂解可导致组织损伤；然而，加剧炎症也是一种感染。 组织破坏的重要原因。本应用程序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- 信息传递。弓形虫将效应蛋白分泌到宿主细胞质中，以拉拢宿主细胞。弓形虫感染的窝点- 树突状细胞(DC)表现出高运动性，感染的DC促进了跨视网膜上皮细胞的迁移 细胞。中心假设是弓形虫利用免疫细胞，如DC，作为特洛伊木马传播- Nate的眼睛和轮回通过血视网膜屏障。使用弓形虫CRISPR/CAS9损失- 在小鼠的功能筛选中，我们的实验室已经确定了多个似乎影响其能力的弓形虫候选基因 在不影响复制效率的情况下定植眼睛。在第一个目标中，将确认是否击倒 根据CRISPR筛查，与眼睛定植有关的弓形虫候选基因具有 受感染小鼠眼睛定植缺陷。体内寄生虫的复制、传播、定植 眼部和眼部炎症将在基因敲除和野生型寄生虫之间进行比较。 随着时间的推移，VO全身和眼睛成像技术在活体小鼠身上的应用。第二个目的是体外CRISPR/ Cas9功能丧失筛查将用于鉴定介导受感染的白细胞迁移的弓形虫基因 穿过极化的人视网膜上皮细胞。对于在目标1中产生的寄生虫敲除菌株，它也将是 测试他们是否在使用DC穿越极化的视网膜上皮细胞方面存在缺陷。拟议的研究是 重要的是因为新的弓形虫基因的识别决定了眼睛和眼部疾病的定植 EASE有可能帮助合理设计治疗眼弓形体病的新疗法。