Invasion of C. neoformans into brain endothelial cells

描述(申请人提供)：致病酵母菌新生隐球菌可通过血液传播并引起破坏性脑膜炎。目前，隐球菌性脑膜炎是最常见的中枢神经系统真菌感染，也是艾滋病患者最常见的神经系统并发症。新生葡萄球菌穿越血脑屏障(BBB)导致中枢神经系统感染的机制目前尚不清楚。该项目的目标是继续研究新生葡萄球菌如何进入构成血脑屏障的人脑微血管内皮细胞(HBMEC)。在上一次资助期间，我们发现新生隐球菌能通过细胞骨架重组诱导HBMEC的形态改变。我们证明了新生隐孢子菌CPS1编码透明质酸合成酶。我们还使用体外BBB模型验证了CPS1是新生葡萄球菌与HBMEC结合所必需的。此外，我们的研究表明，CD44是HBMEC上促进新生葡萄球菌黏附的主要受体。当新生芽孢杆菌与HBMEC结合后，宿主CD44移位到膜筏上并包围酵母进入部位。无论是新生葡萄球菌中CPS1的缺失还是HBMEC上的CD44基因敲除，都显著地削弱了酵母的感染。我们还观察到，在经Filipin、GF109203X、Cytochallasin D处理的HBMEC中，酵母结合和/或侵袭显著减少。Filipin提取膜筏上的胆固醇和小窝蛋白，GF109203X是蛋白激酶C(PKC)抑制剂，细胞松弛素D是F-肌动蛋白干扰试剂。显性负性PKCA也能抑制酵母对HBMEC的侵袭。结果表明，膜筏的完整性、功能性的PKCA和F-肌动蛋白是酵母入侵所必需的。基于以上观察，我们推测酵母进入HBMEC需要CD44诱导的信号和诱导的细胞骨架重组。我们将从以下几个方面探讨新生隐球菌侵袭的机制：(1)确定CD44在新生隐球菌侵袭过程中的信号转导；(2)研究新生隐球菌如何诱导HBMEC上的细胞骨架重组及其与酵母感染的关系；(3)评价CD44在新生隐球菌侵袭小鼠模型中的作用。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。在此授权期内，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，并最终死于这种病原体。从这些研究中获得的信息有望有助于制定新的策略来预防隐球菌性脑膜炎及其相关发病率。公共卫生相关性：该项目的目标是继续研究新生葡萄球菌如何侵入构成血脑屏障的人脑微血管内皮细胞(HBMEC)。在上一次授权期间，我们鉴定并鉴定了一种新的毒力因子CPS1，它是与HBMEC黏附所必需的。我们还证明了宿主CD44是其黏附的主要受体。在这项赠款提案中，我们将探索新生葡萄球菌是如何内化到HBMEC的。从这些研究中获得的信息有望有助于制定新的策略来预防隐球菌性脑膜炎及其相关发病率。

DESCRIPTION (provided by applicant): Pathogenic yeast Cryptococcus neoformans can disseminate through the blood stream and cause devastating meningitis. At present, cryptococcal meningitis is the most common fungal infection of the central nervous system (CNS) and also the most frequent neurological complication in AIDS patients. The mechanism that transversal of C. neoformans across the blood-brain barrier (BBB) to cause the 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 an 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 signals 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 associate morbidity. PUBLIC HEALTH RELEVANCE: The goal of this project is to continue investigating how C. neoformans invades into human brain microvascular endothelial cells (HBMEC), which constitute the blood-brain barrier. In the last grant period, we identified and characterized a novel virulence factor CPS1, which is required for the adhesion to the HBMEC. We also demonstrated that host CD44 is the primary receptor for its adhesion. In this grant proposal, we will explore how C. neoformans internalizes into the HBMEC. 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.