Invasion of C. neoformans into brain endothelial cells

描述（由申请人提供）：致病性酵母菌新型隐球菌可以通过血液传播并引起毁灭性的脑膜炎。隐球菌性脑膜炎是目前艾滋病患者中最常见的中枢神经系统真菌感染，也是最常见的神经系统并发症。新生弓形虫横过血脑屏障（BBB）引起中枢神经系统感染的机制在很大程度上是未知的。该项目的目标是继续研究新生弓形虫如何进入构成血脑屏障的人脑微血管内皮细胞（HBMEC）。在上一个资助期，我们发现新生C.可以通过细胞骨架重组诱导HBMEC的形态学改变。我们证明了新形C. CPS1编码透明质酸合成酶。我们还通过体外血脑屏障模型验证了CPS1是新生c结合HBMEC所必需的。此外，我们的研究表明，CD44是新生c粘附在HBMEC上的主要受体。当新生酵母与HBMEC结合时，宿主CD44转移到膜筏上并包围酵母进入位点。无论是新生C.的CPS1缺失，还是HBMEC的CD44敲除，都能显著降低酵母菌感染。我们还观察到，在filipin-、GF109203X-、细胞松弛素D-处理的HBMEC中，酵母的结合和/或侵袭显著减少。Filipin在膜筏上提取胆固醇和小窝蛋白，GF109203X是一种蛋白激酶C （PKC）抑制剂，细胞松弛素D是一种f -肌动蛋白破坏试剂。显性阴性PKCa也能抑制酵母侵入HBMEC。结果表明，膜筏的完整性、功能性PKCa和f -肌动蛋白是酵母入侵的必要条件。基于上述观察，我们假设cd44引发的信号和诱导的细胞骨架重组是酵母进入HBMEC所必需的。我们将通过以下目的来探索新形态C.入侵的机制：(1)确定新形态C.入侵过程中CD44引发的信号传导；(2)研究新形态C.诱导HBMEC细胞骨架重组及其与酵母菌感染的关系；(3)在小鼠模型中评估CD44在新形态C.入侵中的作用。在我们之前的资助期间，我们已经证明了病原体-宿主相互作用的粘附步骤。在此资助期内，我们将进一步表征内化步骤中的分子事件。这些研究与临床观察有关，大量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预防隐球菌脑膜炎及其相关发病率的新策略。公共卫生相关性：该项目的目标是继续研究新生C.如何侵入构成血脑屏障的人脑微血管内皮细胞（HBMEC）。在上一个资助期，我们鉴定并鉴定了一种新的毒力因子CPS1，这是HBMEC粘附所必需的。我们还证明宿主CD44是其粘附的主要受体。在这项拨款提案中，我们将探讨C. neoformans如何内化到HBMEC中。从这些研究中获得的信息有望有助于制定预防隐球菌脑膜炎及其相关发病率的新策略。

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.