The role of the brain endothelium in fungal infections of the CNS

描述（由申请人提供）：我们对致命真菌病原体（如新型隐球菌）如何侵入中枢神经系统（CNS）的理解存在重大空白。这一差距的持续存在导致了我们对中枢神经系统真菌疾病掌握的重大缺陷，并导致了开发更有效和创新治疗方案的不足。目前关于新型隐球菌脑发病机制的证据表明，隐球菌主要通过脑内皮（又称血脑屏障）进行跨细胞迁移；但促进这一过程的脑内皮因子和关键毒力真菌因子的身份在很大程度上是未知的。在这个特殊的应用中，目的是了解一种新发现的、分泌的真菌金属蛋白酶如何靶向脑内皮蛋白，以及这种关联如何改变真菌-脑内皮界面内的微环境，以促进真菌细胞向中枢神经系统的迁移。核心假设是一种新发现的分泌金属蛋白酶（Mpr1）通过其蛋白水解活性揭开/刺激表面受体/配体和/或粘附蛋白，从而刺激隐球菌向中枢神经系统的迁移。这一假设的提出是基于申请人实验室提供的强有力的初步数据。这项研究的基本原理是，了解诱导新生隐球菌大脑发病机制的分子机制，将转化为开发先进的抗感染药物和疫苗，这些药物和疫苗可能在治疗隐球菌疾病方面非常有效，隐球菌疾病每年夺去约60万人的生命。在强有力的初步数据的指导下，这一假设将通过追求一个特定的目标来验证：通过识别在真菌-脑内皮界面转化表面微环境的Mpr1的宿主靶标来解决脑内皮在新生c向中枢神经系统迁移中的作用。在这里，我们将验证Mpr1直接靶向脑内皮表面蛋白的预测，作为重新定义真菌-脑内皮微环境的一种手段，以支持和促进真菌入侵中枢神经系统。我们将首先通过在非致病性酵母中表达Mpr1来确定Mpr1是否足以附着在脑内皮上，并利用这种表型来识别Mpr1的宿主靶点。其次，我们将使用一个强大的，高含量的蛋白质组学筛选来鉴定Mpr1的天然宿主底物。为了优先考虑和确定这些靶点的作用，将利用血脑屏障的体外模型、隐球菌疾病的体内模型和实时活体显微镜。该研究的创新之处在于其使用体外血脑屏障模型和最先进的方法来研究新形态C.蜂拥中枢神经系统的新机制。我们期望这项研究将促进我们对脑内皮在中枢神经系统真菌疾病中的作用的理解。最终，这些知识有可能为真菌性脑膜脑炎患者的药物和疫苗开发策略注入活力。

DESCRIPTION (provided by applicant): There is a critical gap in our understanding of how a deadly fungal pathogen such as Cryptococcus neoformans invades the central nervous system (CNS). The continued existence of this gap has resulted in a major deficit in our grasp of fungal disease of the CNS and has led to a shortfall in the development of more effective and innovative treatment options. Current evidence on cerebral pathogenesis of C. neoformans has shown a predominantly transcellular migration of cryptococci across the brain endothelium (a.k.a. the blood- brain barrier); but the identities of brain endothelium factors and key virulenc fungal factors that promote this process are largely unknown. The objective in this particular application is to understand how a newly identified, secreted fungal metalloprotease targets brain endothelial proteins and how this association transforms the microenvironment within the fungal-brain endothelium interface to promote the migration of fungal cells into the CNS. The central hypothesis is that a newly identified secreted metalloprotease (Mpr1) stimulates the transmigration of cryptococci into the CNS by unmasking/stimulating surface receptors/ligands and/or adhesion proteins via its proteolytic activity. The formulation of this hypothesis is based on strong preliminary data produced from the laboratory of the applicant. The rationale for the proposed research is that understanding the molecular mechanisms that induce cerebral pathogenesis of C. neoformans will translate into the development of advanced antinfective drugs and vaccines that could be highly effective in the treatment of cryptococcal disease which claims the lives of ~600,00 individuals per year. Guided by strong preliminary data, this hypothesis will be tested by pursuing one specific aim: Resolve the role of the brain endothelium in the transmigration of C. neoformans into the CNS by identifying the host targets of Mpr1 that transform the surface microenvironment at the fungal-brain endothelium interface. Here we will test the prediction that Mpr1 directly targets surface proteins of the brain endothelium as a means to redefine the microenvironment of the fungal-brain endothelium to favor and promote fungal invasion of the CNS. We will do this by first establishing whether Mpr1 is sufficient for attachment to the brain endothelium by expressing Mpr1 in a non-pathogenic yeast and exploiting this phenotype to identify host targets of Mpr1. Secondly we will use a powerful, high-content proteomic screen to identify native host substrates of Mpr1. To prioritize and resolve the role of these targets, an in vitro model of the BBB, in vivo models of cryptococcal disease and real-time intravital microscopy will be utilized. The innovation of the proposed study lies in its use of an in vitro model of the BBB and state-of-the-art methodology to examine a novel mechanism utilized by C. neoformans to swarm the CNS. We anticipate that the proposed study will advance our understanding of the role of the brain endothelium in fungal disease of the CNS. Ultimately such knowledge has the potential to invigorate drug and vaccine development strategies for individuals suffering from fungal meningoencephalitis.