Invasion of C. neoformans into brain endothelial cells

新型隐球菌侵入脑内皮细胞

• 批准号: 8094247
• 负责人: AMBROSE Y JONG
• 金额: $ 39.2万
• 依托单位: CHILDREN'S HOSPITAL OF LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-01 至 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8094247
• 关键词: Abbreviations; Acquired Immunodeficiency Syndrome; Actins; Adhesions; Animals; Applications Grants; Attenuated; Binding; Blood; Blood - brain barrier anatomy; Brain; CD44 gene; CYP21A2 gene; Caveolins; Central Nervous System Fungal Infections; Central Nervous System Infections; Cerebrospinal Fluid; Cholesterol; Clinical; Colony-forming units; Complication; Cryptococcal Meningitis; Cryptococcus neoformans; Cytochalasin D; Cytoskeleton; Development; Dominant-Negative Mutation; Electron Microscope; Endothelial Cells; Event; F-Actin; Filipin; Goals; Grant; Health; Human; Hyaluronan; Hyaluronic Acid; Immune response; In Vitro; Infection; Invaded; Knock-out; Knockout Mice; Membrane; Meningitis; Modeling; Molecular; Morbidity - disease rate; Mus; Mutation; Neuraxis; Neurologic; Patients; Process; Protein Kinase C; Protein Kinase C Inhibitor; Reagent; Role; Scanning; Severities; Signal Transduction; Site; Stream; Testing; Virulence; Virulence Factors; Yeasts; base; caveolin 1; cellular microvillus; citrate carrier; mouse model; novel; novel strategies; pathogen; prevent; receptor; transmission process

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.

描述（由申请人提供）：致病性酵母菌新型隐球菌可通过血流传播并引起毁灭性脑膜炎。隐球菌脑膜炎是目前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。探讨了C.新生儿通过血脑屏障（BBB）引起CNS感染的可能性在很大程度上是未知的。本项目的目标是继续研究C。新生儿进入人脑微血管内皮细胞（HBMEC），其构成BBB。在上一个资助期，我们发现C。新生儿可通过细胞骨架重组引起HBMEC的形态学改变。我们证明了C.新生儿CPS 1编码透明质酸合成酶。我们还验证了CPS 1是C.使用体外BBB模型测定新生儿与HBMEC的结合。此外，我们的研究表明，CD 44是C.新生儿粘连。在C。当新生儿与HBMEC结合时，宿主CD 44易位至膜筏并包围酵母进入位点。C.新生儿或CD 44敲除的HBMEC显著削弱了酵母菌感染。我们还观察到，在菲律宾肽、GF 109203 X、细胞松弛素D处理的HBMEC中，酵母结合和/或侵袭显著降低。Filipin提取膜筏上的胆固醇和小窝蛋白，GF 109203 X是蛋白激酶C（PKC）抑制剂，而细胞松弛素D是F-肌动蛋白破坏剂。显性负性PKCa也抑制酵母侵入HBMEC。结果表明，膜筏的完整性，功能性的PKCa和F-肌动蛋白是必要的酵母入侵。基于上述观察，我们假设CD 44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。我们将探讨C.（1）研究CD 44在新生隐球菌侵袭过程中的作用。（2）探讨C.探讨新生隐球菌感染对HBMEC细胞骨架的影响及其与酵母菌感染的关系。小鼠模型中的新生动物侵袭。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。在这一资助期内，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，即大量患者患有严重的脑膜炎并最终死于这种病原体。从研究中获得的信息有望有助于开发新的策略来预防隐球菌脑膜炎及其相关的发病率。公共卫生相关性：本项目的目标是继续调查C。新生儿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CPS 1，这是粘附HBMEC所必需的。我们还证明了宿主CD 44是其粘附的主要受体。在这篇论文中，我们将探讨C。新生儿内化到HBMEC中。从研究中获得的信息预计将有助于开发新的策略，以预防隐球菌脑膜炎及其相关的发病率。