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， 与中枢神经系统感染的其他病原体相比，死亡率。新生隐球菌 (CN)、白色念珠菌、曲霉菌、毛霉、球孢子虫和组织胞浆菌 是导致脑部真菌感染的最常见原因，导致慢性而不是急性 或亚急性脑膜脑炎。长期目标将研究时空 跨血脑屏障贩运CN的组织和分子基础，并调查串扰 跨细胞机制和对CN的反应打开细胞旁通路之间的关系 感染。根据我们的初步数据和已发表的研究，我们认为CN代表一种 极佳的模式病原体，用来确定病原真菌用来 突破BBB，进入CNS。在我们努力理解调节分子基础的过程中 在CN-脑内皮细胞的相互作用中，我们发现了一个酪氨酸激酶受体(EphA2-TKR)，它是 中锋球员。中心假说认为，真菌细胞通过参与血脑屏障 EphA2-TKR，以便访问进入CNS的二进制路径。为了检验我们的假设，我们将 研究真菌细胞、EphA2-TRK及其下游信号的相互作用 介导CN从血到脑转运的成分。以下是具体目标 将检验假设：SA1将调查CN诱导的CD44介导的 EphA2-TKR活性及EphA2-TKR的相互作用SA2将研究EphA2-TKR的作用 在上调CN的囊泡交通和解决CN是否诱导EphA2-TKR活性方面 促进跨细胞和旁细胞途径的共同调节。SA3将解决 EphA2-TKR活性在体内CN从血到脑转运中的作用 研究CN攻击时血脑屏障通透性变化的分子基础。 这项拟议的研究具有创新性，因为它将首次详细说明 促进整个生物圈内真菌的内化和贩运。这项创新延伸到我们的 已发表的研究发现，脑内皮细胞中的EphA2-TKR是 调节CN从血液到脑的迁移。此外，还有一种观点认为， BBB是由真菌诱导的CD44-EphA2-TKR反式激活来触发病理的 脑内皮细胞的内吞作用(以及可能的次级细胞旁途径)的使用是一种 从我们目前的理解转变为范式。我们提出了一些实验，这些实验将使用 多种互补方法梳理CN、EphA2-之间的分子相互作用 TKR和CD44，同时也使用邻近依赖标记来鉴定相互作用组 EphA2-TKR。我们从研究中获得的知识将对真菌研究做出重大贡献 中枢神经系统的发病机制，因为它仍然被严重地研究不足，这项工作将 带头开发可以阻止真菌进入的分子，从而作为前驱 对有发生真菌性脑膜脑炎风险的患者进行预防性或预防性治疗。

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.