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：
10079284
负责人：
Brendan Cormack
金额：
$ 24.56万
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 2022-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10079284
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.

促成血脑屏障渗透的Neoformans因素抽象的 Neoformans的隐孢子会引起威胁生命的中枢神经系统（CNS）感染免疫功能低下的个体，例如HIV-1感染的患者，每年导致超过18万人死亡。新生梭菌的感染开始于从环境中吸入真菌细胞，其次是肺外扩散，导致血源性向目标器官传播，最常见导致中枢神经系统感染。人类病例和实验动物模型的几条证据。 Neoformans CNS感染表明，循环新甲状腺癌最初通过脑毛细血管。由于脑链球菌进入大脑的脑膜杆菌发生在脑微脉管系统中，我们其他人则将体外血脑屏障模型与人脑微血管内皮细胞使用（HBMEC）研究血脑屏障的Neofors渗透。我们证明了新生虫菌株在体外表现出横穿HBMEC单层并在体内渗透到大脑中的能力实验性血源性C. Neoformans CNS感染的小鼠模型，但潜在的机制保持不完全理解。我们和其他人表明，新甲状腺菌刺激了加密局因子的因素血脑屏障的渗透，但确定此类因素仍然不完整。我们生成了来自C. Neoformans基因组DNA的基因组尺度SGRNA库。每个sgrna盒都用作遗传突变跟踪的条形码，可以通过深度测序进行高通量屏幕。我们假设这一点该库允许全基因组筛选出促进血脑的新生虫因子。障碍。通过我们的体外鉴定加密噬菌因子，这一假设得到了支持在HBMEC单层中的测定）和体内血脑屏障模型（隐球菌的动物模型静脉内和鼻内接种后，渗透到大脑中）。我们与两个敲除突变体表明，这种新梭菌因子有助于血液渗透体外和体内脑屏障。这些发现表明，从我们的基因组中鉴定出的新生孢子因素宽屏方法可能有助于血脑屏障的渗透，这是开发Neoformans CNS感染。该应用程序的创新方面是研究C. Neoformans通过阐明新鉴定的加密秒因子来穿透血脑屏障。这从该应用程序得出的信息将为研究C的发病机理提供新的范式。 Neoformans CNS感染。