Invasion of C. neoformans into brain endothelial cells

致病酵母菌新生芽孢杆菌可以通过血液传播，并造成毁灭性的 脑膜炎。新生葡萄球菌穿越血脑屏障(BBB)导致脑出血的机制 中枢神经系统(CNS)感染在很大程度上是未知的。这个项目的目标是继续 研究新生葡萄球菌是如何进入人脑微血管内皮细胞的 构成了血脑屏障。 在上一次资助期间，我们发现新生隐球菌能诱导HBMEC发生形态变化。 通过细胞骨架重组。我们证明了新生葡萄球菌cps1编码透明质酸。 合成酶。我们还通过体外实验验证了CPS1是新生葡萄球菌与HBMEC结合所必需的。 BBB模型。此外，我们的研究表明，CD44是C。 新生杆菌粘连。新生芽孢杆菌与HBMEC结合后，宿主CD44移位到HBMEC 膜排，并包围了酵母进入部位。新生葡萄球菌中的CPS1缺失或CD44- HBMEC基因敲除可显著降低酵母菌感染。我们还观察到酵母结合和/或 经FIIPIN、GF109203X、细胞松弛素D处理的HBMEC侵袭力显著降低。过滤 提取膜筏上的胆固醇和小窝蛋白，GF109203X是一种蛋白激酶C(PKC)抑制剂， 而细胞松弛素D是一种F-肌动蛋白干扰剂。显性负PKCA也抑制了酵母的入侵 进入HBMEC。结果表明，膜筏的完整性、功能性PKCA和F-肌动蛋白 对于酵母菌入侵是必要的。根据以上观察结果，我们推测CD44- 酵母进入HBMEC需要信号转导和诱导的细胞骨架重组。我们将探索 新生隐孢子虫的侵袭机制主要有以下几个方面：(1)检测CD44诱导的CD44 新生链球菌入侵过程中的信号传递，(2)研究新生链球菌如何诱导细胞骨架 重组HBMEC及其与酵母感染的关系；(3)评价CD44的作用 在新生芽孢杆菌入侵小鼠模型期间。 在我们之前的授权期，我们已经演示了病原体-宿主相互作用的黏附步骤。在……里面 在这个授权期内，我们将进一步描述内化步骤中的分子事件。这些研究 与临床观察有关，即相当数量的患者患有严重的脑膜炎和 最终死于这种病原体。从研究中获得的信息预计将有助于 预防隐球菌性脑膜炎及其相关发病率的新策略的发展。

Pathogenic yeast C. neoformans can disseminate through the blood stream and cause devastating meningitis. The mechanism transversal of C. neoformans across the blood-brain barrier (BBB) to cause the central nervous system (CNS) infection is largely unknown. The goal of this project is to continue investigating how C. neoformans enters into human brain microvascular endothelial cells (HBMEC), which constitute the BBB. In the last grant period, we found that C. neoformans could induce morphological changes in HBMEC via cytoskeleton reorganization. We demonstrated that C. neoformans CPS1 encoded hyaluronic acid synthase. We also verified that CPS1 was required for C. neoformans binding to HBMEC using an in vitro BBB model. Furthermore, our studies showed that CD44 was the primary receptor on HBMEC for C. neoformans adhesion. Upon C. neoformans binding to the HBMEC, host CD44 translocated to the membrane rafts and surrounded the yeast entry site. Either CPS1 deletion in C. neoformans or CD44- knockout on HBMEC significantly impaired the yeast infection. We also observed that yeast binding and/or invasion was considerably reduced in the filipin-, GF109203X-, cytochalasin D- treated HBMEC. Filipin extracts cholesterol and caveolin on the membrane rafts, GF109203X is a Protein Kinase C (PKC) inhibitor, and cytochalasin D is a F-actin disrupting reagent. Dominant-negative PKCa also inhibited yeast invasion into HBMEC. The results suggest that the integrity of membrane rafts, functional PKCa, and F-actin were necessary for yeast invasion. Based on the above observations, we hypothesize that CD44-elicited signaling and induced cytoskeleton reorganization are required for yeast entry into HBMEC. We will explore the mechanisms of C. neoformans invasion by the following Aims: (1) To determine the CD44-elicited signaling during the C. neoformans invasion, (2) To examine how C. neoformans induces cytoskeleton reorganization on HBMEC and its relationship to yeast infection, and (3) To evaluate the role of CD44 during C. neoformans invasion in mouse models. In our previous grant period, we have demonstrated the adhesion step of pathogen-host interaction. In this grant period, we will further characterize the molecular events at the internalization step. These studies are related to the clinical observations that a significant number of patients suffer severe meningitis and eventually succumb to this pathogen. The information derived from the studies is expected to be helpful in the development of novel strategies to prevent cryptococcal meningitis and its associated morbidity.