The role of inositol in Cryptococcus biology and pathogenesis

肌醇在隐球菌生物学和发病机制中的作用

• 批准号: 9903576
• 负责人: Chaoyang Xue
• 金额: $ 8.72万
• 依托单位: RBHS-NEW JERSEY MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-11-24 至 2021-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9903576
• 关键词: Anabolism; Animal Model; Antifungal Agents; Binding; Biological Assay; Biological Models; Biology; Biotin; Blood; Blood - brain barrier anatomy; Blood Vessels; Brain; Cell surface; Cells; Central Nervous System Infections; Cessation of life; Complex; Cryptococcal Meningitis; Cryptococcus; Cryptococcus neoformans; Development; Disease; Endothelium; Environment; Exhibits; Extravasation; Exudate; Fungal Meningitis; Future; Gene Deletion; Genetic Transcription; Goals; Growth; Heart Transplantation; Hemostatic function; Homeostasis; Human; Hyaluronic Acid; Immune Evasion; Immunity; Immunocompromised Host; In Vitro; Infection; Inositol; Knowledge; Label; Laboratories; Lead; Life; Ligands; Mediating; Meningitis; Metabolic Pathway; Metabolism; Mission; Modeling; Molecular; Mus; Mutagenesis; Mycoses; Neuraxis; Neurotropism; Pathogenesis; Pathway interactions; Plants; Play; Polysaccharides; Process; Production; Quantitative Reverse Transcriptase PCR; Role; Structure; System; Testing; Tight Junctions; Transplant Recipients; United States National Institutes of Health; Virulence; Virulence Factors; base; blood-brain barrier crossing; blood-brain barrier penetration; capsule; chromatin immunoprecipitation; deletion library; disorder control; disorder prevention; fungus; high throughput screening; human pathogen; in vivo; in vivo imaging system; insight; monolayer; mutant; nano-string; novel; novel strategies; pathogen; pathogenic fungus; promoter; transcription factor; transcriptome sequencing; uptake

ABSTRACT Cryptococcus neoformans is a deadly fungal pathogen that exhibits pronounced neurotropism: it is the most common cause of fungal meningitis, particularly in immunocompromised patients, resulting in over 620,000 deaths annually. How C. neoformans cells traverse the blood-brain barrier (BBB) to infect the central nervous system (CNS) remains poorly understood. Our recent studies implicate inositol – one of the most abundant metabolites in the brain – in this process. In particular, we find that growth of C. neoformans under inositol-rich conditions enhances fungal virulence and that fungal mutants defective in inositol uptake exhibit reduced virulence, reduced capacity to transmigrate from the blood into the brain, and reduced ability to traverse a model BBB in vitro. We also find that C. neoformans compromises tight-junction integrity in vitro, promoting inositol leakage through the brain microvascular endothelial monolayer. Furthermore, we show that inositol induces the expression of fungal cell surface factors involved in virulence, including the polysaccharide capsule, a major fungal virulence factor, and hyaluronic acid (HA), a ligand important for fungal binding to the BBB. Finally, we find that growth on inositol promotes the production of capsule structures involved in immune evasion and that, conversely, C. neoformans mutants defective in inositol uptake elicit enhanced protective immunity during brain infection. Based on these results, we hypothesize that C. neoformans senses and utilizes host inositol to modify the fungal cell surface in a way that promotes penetration of the BBB and development of cryptococcal meningitis. Interestingly, C. neoformans contains an unusually complex inositol uptake system and catabolic pathway, which likely evolved from its utilization of the inositol stores of its plant reservoirs. Thus, this fungus may be uniquely adapted to thrive in the inositol-rich environment of the CNS and to utilize inositol-dependent pathways for pathogenesis. The overarching goal of this proposal is to obtain a detailed understanding of the mechanism by which C. neoformans acquires and utilizes host inositol to establish human brain infection. We propose three Specific Aims: 1) Define inositol sensing and metabolic pathways required for modifying fungal cell surface structure by using a combination of fungal mutagenesis analysis, enzymatic assays, and polysaccharide structural analysis; 2) Characterize the mechanism of inositol promotion in C. neoformans BBB crossing and CNS infection by using an in vitro BBB model system and in vivo animal models; and 3) Define the transcriptional circuits regulating inositol-dependent processes during cryptococcal brain infection by analyzing the expression and localization of fungal inositol factors and identifying transcription factors regulating their expression during infection. Together, these studies will elucidate a novel contribution of a brain metabolite, inositol, to the development of life-threatening fungal meningitis. As such, these studies promise to provide substantial insight into mechanisms by which pathogens cross the BBB and establish CNS infections.

摘要 新型隐球菌是一种致命的真菌病原体，表现出明显的嗜神经性：它是最 真菌性脑膜炎的常见原因，特别是在免疫功能低下的患者中，导致超过620，000 每年死亡。如何C。新生儿细胞穿过血脑屏障（BBB）感染中枢神经系统， 系统（CNS）仍然知之甚少。我们最近的研究表明肌醇是最丰富的 大脑中的代谢物-在这个过程中。特别地，我们发现C.富含肌醇的新生儿 条件增强真菌毒力，并且肌醇摄取缺陷的真菌突变体表现出降低的 毒力，降低从血液进入大脑的能力，以及降低穿越大脑的能力。 体外BBB模型。我们还发现C.新生儿在体外损害紧密连接的完整性， 肌醇通过脑微血管内皮单层渗漏。此外，我们表明，肌醇 诱导参与毒力的真菌细胞表面因子的表达，包括多糖 囊，一种主要的真菌毒力因子，和透明质酸（HA），一种对真菌结合到 BBB.最后，我们发现在肌醇上的生长促进了参与免疫反应的胶囊结构的产生。 回避，反之，C.肌醇摄取缺陷的新型变形杆菌突变体引起增强的保护性 大脑感染时的免疫力基于这些结果，我们假设C。neoformans感官和 利用宿主肌醇以促进BBB渗透的方式修饰真菌细胞表面， 隐球菌脑膜炎的发展。有趣的是，C。neoformans含有一种异常复杂的肌醇 吸收系统和分解代谢途径，这可能是从其利用其植物的肌醇储存演变而来的 水库因此，这种真菌可能是唯一适合在中枢神经系统富含肌醇的环境中生长的真菌， 利用肌醇依赖性途径致病。该提案的总体目标是获得一个 详细了解了C.新生儿获得并利用宿主肌醇， 建立人类大脑感染我们提出了三个具体的目标：1）定义肌醇传感和代谢 通过使用真菌诱变组合修饰真菌细胞表面结构所需的途径 分析，酶法测定和多糖结构分析; 2）表征肌醇的作用机理 在C.通过使用体外血脑屏障模型系统和在 体内动物模型;和3）定义转录电路调节肌醇依赖性过程中， 通过分析真菌肌醇因子的表达和定位， 鉴定在感染期间调节其表达的转录因子。这些研究将 阐明脑代谢产物肌醇对威胁生命的真菌发展的新贡献 脑膜炎因此，这些研究有望为病原体 穿过BBB并建立中枢神经系统感染。