The molecular basis for the translocation of fungi from blood-to-brain.

项目摘要 真菌引起的中枢神经系统（CNS）感染发病率最高， 与CNS感染的其他病原体相比，新生隐球菌 (Cn)，白色念珠菌，曲霉属，毛霉属，球孢子菌属，和荚膜组织胞浆菌 是导致慢性而不是急性脑真菌感染的最常见原因 或亚急性脑膜脑炎。长期目标是调查 的组织和分子基础的Cn运输通过血脑屏障和调查的串扰 在跨细胞机制和响应Cn的细胞旁通路的打开之间 感染根据我们的初步数据和已发表的研究，我们提出Cn代表一个 很好的模式病原体，以确定致病真菌使用的机制， 突破血脑屏障进入中枢神经系统在我们努力了解介导 在Cn-脑内皮相互作用中，我们鉴定了酪氨酸激酶受体（EphA 2-TKR）， 中心球员。中心假设指出，真菌细胞通过与血脑屏障结合而穿透血脑屏障。 EphA 2-TKR，以便访问进入CNS的二元路径。为了验证我们的假设，我们将 研究真菌细胞、EphA 2-TRK及其下游信号传导之间的相互作用 在介导Cn从血液到脑的易位中的组分。以下具体目标 将检验假设：SA 1将研究Cn诱导的CD 44介导的 EphA 2-TKR活性和EphA 2-TKR的相互作用组。SA 2将检查EphA 2-TKR的作用 在上调Cn的囊泡运输和解决Cn是否诱导EphA 2-TKR活性方面， 促进跨细胞和细胞旁途径的共调节。SA 3将解决 EphA 2-TKR活性对体内Cn从血液到脑的易位的贡献， 检查当受到Cn挑战时BBB渗透性变化的分子基础。 拟议的研究是创新的，因为它将首次详细介绍 促进真菌穿过BBB的内化和运输。创新延伸到我们的 已发表的研究表明，脑内皮中的EphA 2-TKR是脑血管病的核心成分。 介导Cn从血液到脑的迁移。这沿着的概念是， BBB是由真菌诱导的EphA 2-TKR的CD 44反式激活引发的，以触发病理性的 在脑内皮细胞中使用内吞作用（可能是次要的细胞旁途径）是一种 从我们目前的理解模式转变。我们已经提出了实验， 多种互补的方法来梳理Cn、EphA 2-EphA 3之间的分子相互作用， TKR和CD 44的相互作用，同时也使用邻近依赖性标记来鉴定TKR和CD 44的相互作用组。 EphA2-TKR。从我们的研究中获得的知识将对真菌学做出重大贡献。 中枢神经系统的发病机制，因为它仍然显着研究不足，这项工作将 率先开发能够阻止真菌进入的分子，从而作为预防真菌入侵的手段。 在有发生真菌性脑膜脑炎风险的患者中进行预防性治疗。

PROJECT ABSTRACT Infections of the central nervous system (CNS) caused by fungi have the highest morbidity and mortality when compared to other causative agents of CNS infections. Cryptococcus neoformans (Cn), Candida albicans, Aspergillus spp., Mucor, Coccidioides spp., and Histoplasma capsulatum are the most common cause of fungal brain infections resulting in a chronic instead of an acute or subacute meningoencephalitis. The long-term goal will investigate the spatio-temporal organization and molecular basis of Cn trafficking across the BBB and investigate a cross-talk between a transcellular mechanism and the opening of a paracellular path in response to Cn infection. Given our preliminary data and published studies, we propose that Cn represents an excellent model pathogen with which to determine the mechanisms that pathogenic fungi use to breach the BBB and enter the CNS. In our efforts to understand the molecular basis mediating the Cn-brain endothelium interactions, we identified a tyrosine kinase receptor (EphA2-TKR) as the central player. The central hypothesis states that fungal cells penetrate the BBB by engaging EphA2-TKR in order to access a binary path into the CNS. To test our hypothesis, we will investigate the interplay between fungal cells, EphA2-TRK and its downstream signaling components in mediating the translocation of Cn from blood-to-brain. The following specific aims will test the hypothesis: SA1 will investigate a Cn-induced CD44-mediated transactivation of EphA2-TKR activity and the interactome of EphA2-TKR. SA2 will examine the role of EphA2-TKR in upregulating vesicular traffic of Cn and resolve whether Cn-induced activity of EphA2-TKR promotes a co-regulation of a transcellular and paracellular pathway. SA3 will resolve the contribution of EphA2-TKR activity to the translocation of Cn from blood-to-brain in vivo and examine the molecular basis of changes in the permeability of the BBB when challenged by Cn. The proposed research is innovative because it will for the first time, detail the key players that promote the internalization and trafficking of fungi across the BBB. The innovation extends to our published studies that identified EphA2-TKR in the brain endothelium as a central element in mediating the migration Cn from blood-to-brain. This along with the notion that crossing of the BBB is initiated by a fungal-induced CD44-transactivation of EphA2-TKR to trigger a pathological use of endocytosis (and possibly a secondary paracellular path) in brain endothelial cells is a paradigm shift from our current understanding. We have proposed experiments that will use multiple complementary approaches to tease apart the molecular interaction between Cn, EphA2- TKR and CD44 while also using proximity-dependent labeling to identify the interactome of EphA2-TKR. The knowledge gained from our studies will make a significant contribution to fungal pathogenesis of the CNS, because it remains significantly understudied, and this work will spearhead the development of molecules that could block fungal entry and thus serve as pre- emptive or prophylactic therapy in patients at risk for developing fungal meningoencephalitis.