The role of inositol in Cryptococcus biology and pathogenesis

摘要 新生隐球菌是一种致命的真菌病原体，表现出明显的神经趋向性：它是 真菌脑膜炎的常见原因，特别是在免疫功能低下的患者中，导致超过620,000人 每年的死亡人数。新生芽孢杆菌细胞如何穿越血脑屏障感染中枢神经系统 对中枢神经系统(CNS)仍知之甚少。我们最近的研究涉及肌醇--最丰富的 大脑中的代谢物--在这个过程中。特别是，我们发现新生隐孢子虫在富含肌醇的环境中生长 条件增强真菌毒力，摄取肌醇缺陷的真菌突变体表现出减少 毒性，从血液转移到大脑的能力降低，以及穿越 体外模拟血脑屏障。我们还发现，新生葡萄球菌在体外损害了紧密连接的完整性，促进了 肌醇通过脑微血管内皮细胞单层渗漏。此外，我们还证明了肌醇 诱导参与毒力的真菌细胞表面因子的表达，包括多糖 衣壳，一个主要的真菌毒力因子，和透明质酸(HA)，一个重要的配体，真菌结合到 BBB。最后，我们发现，肌醇的生长促进了与免疫有关的胶囊结构的产生。 回避和相反，摄取肌醇缺陷的新生葡萄球菌突变株可诱导增强保护作用 脑部感染时的免疫力。根据这些结果，我们假设新生隐孢子虫感觉和 利用宿主肌醇来修饰真菌细胞表面，以促进BBB的穿透和 隐球菌性脑膜炎的发展。有趣的是，新生葡萄球菌含有一种异常复杂的肌醇。 摄取系统和分解代谢途径，可能是由于其对植物肌醇储备的利用而演变的 水库。因此，这种真菌可能独特地适应在富含肌醇的中枢神经系统和 利用肌醇依赖途径在发病机制中的应用。这项提议的首要目标是获得一个 详细了解新生隐孢子菌获取和利用宿主肌醇的机制 确定人脑感染。我们提出了三个具体目标：1)定义肌醇感觉和代谢 利用真菌诱变组合改变真菌细胞表面结构所需的途径 分析、酶分析和多糖结构分析；2)表征肌醇的作用机理 利用体外BBB模型系统促进新生弧菌BBB交叉和中枢神经系统感染 活体动物模型；以及3)定义调控肌醇依赖过程的转录电路 隐球菌脑感染与真菌肌醇因子的表达和定位分析 确定在感染过程中调节其表达的转录因子。总而言之，这些研究将 阐明脑部代谢物肌醇对威胁生命的真菌的发展的新贡献 脑膜炎。因此，这些研究有望提供对病原体致病机制的实质性见解 越过血脑屏障，确定中枢神经系统感染。

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.