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Cryptococcus neoformans factors contributing to penetration of the blood-brain barrier

新型隐球菌穿透血脑屏障的因素

基本信息

批准号：
10170285
负责人：
Brendan Cormack
金额：
$ 20.47万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-06-01 至 2023-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10170285
关键词：
Agrobacterium Animal Model Bar Codes Bioinformatics Biological Assay Blood - brain barrier anatomy Brain Categories Cells Central Nervous System Infections Cessation of life Code Complement Coupled Cryptococcus Cryptococcus neoformans DNA Defect Development Environment Exhibits Experimental Animal Model Experimental Models Genes Genetic Genome Genomic DNA Goals Growth Guide RNA HIV-1 Hematogenous Human Hyaluronic Acid Immunocompromised Host In Vitro Individual Infection Inhalation Intravenous Kinetics Knock-out Knowledge Libraries Life Ligation Mediating Meningoencephalitis Metalloproteases Methods Modeling Morphology Mutation Organ Parents Pathogenesis Patients Penetration Phospholipase Proteins Public Health RNA library Southern Blotting Urease base blood-brain barrier penetration brain endothelial cell cerebral capillary cerebral microvasculature deep sequencing fungal genetics genome wide screen genome-wide high throughput screening in vivo innovation interest monolayer mouse model mutant novel strategies phosphopantetheinyl transferase transcytosis

项目摘要

Cryptococcus neoformans factors contributing to penetration of the blood-brain barrier Abstract Cryptococcus neoformans causes life-threating central nervous system (CNS) infection in immunocompromised individuals such as HIV-1-infected patients, resulting in over 180,000 deaths annually. Infection with C. neoformans starts with inhalation of fungal cells from environment, followed by extrapulmonary spread, which leads to hematogenous dissemination to target organs, most commonly resulting in CNS infection. Several lines of evidence of human cases and experimental animal models of C. neoformans CNS infection indicate that circulating C. neoformans penetrates into the brain initially via the cerebral capillaries. Since C. neoformans entry into the brain occurred in the cerebral microvasculature, we and others used the in vitro blood-brain barrier model with human brain microvascular endothelial cells (HBMEC) to investigate C. neoformans penetration of the blood-brain barrier. We showed that C. neoformans strains exhibited the ability to traverse the HBMEC monolayer in vitro and penetrate into the brain in vivo in the mouse model of experimental hematogenous C. neoformans CNS infection, but the underlying mechanisms remain incompletely understood. We and others showed that C. neoformans exploits cryptococcal factors for penetration of the blood-brain barrier, but determination of such factors remains incomplete. We generated genome-scale sgRNA library from C. neoformans genomic DNA. Each sgRNA cassette serves as a genetic barcode for mutation tracking, which allows high-throughput screen by deep sequencing. We hypothesize that this library allows genome-wide screen of C. neoformans factors contributing to penetration of the blood-brain barrier. This hypothesis is supported by our identification of cryptococcal factors using our in vitro (transcytosis assays in HBMEC monolayer) and in vivo blood-brain barrier models (animals models of cryptococcal penetration into the brain following intravenous and intranasal inoculations). Our proof of concept study with two knock-out mutants demonstrated that such C. neoformans factors contributed to penetration of the blood- brain barrier in vitro and in vivo. These findings suggest that C. neoformans factors identified from our genome- wide screen method are likely to contribute to penetration of the blood-brain barrier, the essential step in the development of C. neoformans CNS infection. The innovative aspect of this application is to investigate how C. neoformans penetrates the blood-brain barrier by elucidating newly identified cryptococcal factors. The information derived from this application will provide a new paradigm for investigating the pathogenesis of C. neoformans CNS infection.

新生隐球菌穿透血脑屏障的相关因素摘要新型隐球菌引起危及生命的中枢神经系统（CNS）感染，免疫功能低下的个体，如HIV-1感染的患者，每年导致超过180，000人死亡。梭菌感染新生儿首先从环境中吸入真菌细胞，肺外播散，导致血行播散至靶器官，最常见的是导致CNS感染。人类病例和实验动物模型的几条证据表明，新生儿CNS感染表明循环C.新生儿最初是通过脑毛细血管自从C.新生儿入脑发生在脑微血管，我们其他人则使用了人脑微血管内皮细胞的体外血脑屏障模型（HBMEC）对C.新生儿对血脑屏障的渗透我们证明了C.新生菌株表现出在体外穿过HBMEC单层并在体内渗透到脑中的能力。小鼠实验性血源性C.新生儿CNS感染，但其潜在机制仍然不完全理解。我们和其他人证明了C.新形式利用隐球菌因子，血脑屏障的渗透，但这些因素的测定仍然不完整。我们产生来自C.的基因组规模sgRNA文库新生儿基因组DNA每个sgRNA盒作为一个基因表达盒，用于突变跟踪的条形码，其允许通过深度测序进行高通量筛选。我们假设该文库允许C的全基因组筛选。有助于血脑渗透的新生因子屏障这一假设得到了我们使用体外（转胞吞作用）鉴定隐球菌因子的支持在HBMEC单层中的测定）和体内血脑屏障模型（隐球菌感染的动物模型）中进行比较。在静脉内和鼻内接种后渗透到脑中）。我们的概念验证研究，两个基因敲除突变体表明，这种C.新生儿因素有助于血液渗透- 体外和体内脑屏障。这些结果表明C.从我们的基因组中发现的新形式因子宽屏幕方法可能有助于穿透血脑屏障，这是治疗的关键步骤。 C.发展新生儿CNS感染。本申请的创新之处在于研究C. 通过阐明新鉴定的隐球菌因子，新型隐球菌可穿透血脑屏障。的从这一应用中获得的信息将为研究C. 新生儿CNS感染。