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

• 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
• 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

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.

新生隐球菌促进血脑屏障穿透的因素 摘要 新生隐球菌引起危及生命的中枢神经系统(CNS)感染 免疫功能受损的个人，如艾滋病毒-1感染者，每年导致超过18万人死亡。 感染新生隐球菌始于吸入环境中的真菌细胞，其次是 肺外扩散，导致血源性扩散到靶器官，最常见的是 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。 新生杆菌中枢神经系统感染表明循环中的新生杆菌最初通过 脑部毛细血管。由于新生葡萄球菌进入大脑是在脑微血管系统中进行的，我们 另一些人则使用了人脑微血管内皮细胞的体外血脑屏障模型 (HBMEC)，以调查新生葡萄球菌对血脑屏障的渗透。我们证明了新生隐球菌属 菌株在体外显示出穿过HBMEC单层的能力，并在体内穿透到脑内。 实验性血源性新生芽孢杆菌中枢神经系统感染的小鼠模型及其机制 仍然没有完全被理解。我们和其他人表明，新生隐球菌利用隐球菌因子 但对这些因素的确定仍不完整。我们生成了 新生隐翅虫基因组dna的基因组规模sgRNA文库。每个sgRNA盒都充当一个基因 用于突变跟踪的条形码，允许通过深度测序进行高通量筛选。我们假设 该文库可以在全基因组范围内筛选有助于血脑渗透的新生葡萄球菌因子。 障碍。这一假说得到了我们使用体外(细胞转运)鉴定隐球菌因子的支持 HBMEC单层和体内血脑屏障模型(隐球菌动物模型)的检测 在静脉和鼻腔接种后进入大脑)。我们的概念验证研究 两个基因敲除突变体证明了这种新生葡萄球菌因子有助于血液渗透- 体外和体内的脑屏障。这些发现表明，从我们的基因组中识别出的新生葡萄球菌因子- 宽屏幕方法可能有助于穿透血脑屏障，这是 新生葡萄球菌中枢神经系统感染的研究进展这个应用程序的创新之处在于研究C。 新生杆菌通过阐明新发现的隐球菌因子来穿透血脑屏障。这个 从这一应用中获得的信息将为研究C。 新生杆菌中枢神经系统感染。